WO2007052716A1 - Appareil, procede, logiciel et systeme de culture cellulaire - Google Patents

Appareil, procede, logiciel et systeme de culture cellulaire Download PDF

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Publication number
WO2007052716A1
WO2007052716A1 PCT/JP2006/321901 JP2006321901W WO2007052716A1 WO 2007052716 A1 WO2007052716 A1 WO 2007052716A1 JP 2006321901 W JP2006321901 W JP 2006321901W WO 2007052716 A1 WO2007052716 A1 WO 2007052716A1
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WIPO (PCT)
Prior art keywords
culture
cell
cells
bag
medium
Prior art date
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PCT/JP2006/321901
Other languages
English (en)
Japanese (ja)
Inventor
Norihiko Hata
Hidemasa Jinguji
Atsutaka Noguchi
Shiho Sato
Kazutoshi Sato
Naoko Ariyoshi
Akihiro Hosoi
Original Assignee
Medinet Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medinet Co., Ltd. filed Critical Medinet Co., Ltd.
Priority to AU2006309682A priority Critical patent/AU2006309682A1/en
Priority to JP2007542788A priority patent/JP5243038B2/ja
Priority to EP06822821.2A priority patent/EP1944358B1/fr
Priority to US12/084,363 priority patent/US8383395B2/en
Publication of WO2007052716A1 publication Critical patent/WO2007052716A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/58—Reaction vessels connected in series or in parallel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • Cell culture device cell culture method, cell culture program, and cell culture system
  • the present invention relates to a cell culture apparatus, a cell culture method, a cell culture program, and a cell culture system that evaluate cell culture conditions and culture cells.
  • Patent Document 1 grasps the proliferation ability of the entire cell population by observing the morphology of each adhesion-dependent cell without attacking and nondestructively with an image.
  • Patent Document 2 a plurality of casters (containers) are arranged in one thermostat (culture chamber).
  • a cell culturing system is disclosed in which cells are cultivated by storing culture containers one by one in a caster. In this culture system, all the canisters in a single thermostatic chamber have the same culture environment. It has been determined.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2002-218995
  • Patent Document 2 JP-A-2005-73566
  • An object of the present invention has been made in consideration of the above-described circumstances, and a cell culturing apparatus and a cell culturing device capable of realizing appropriate culturing according to the culturing state of the cells while reducing the labor of the operator. It is in providing a cultivation method and a cell culture program.
  • a further object of the present invention is to automatically collect and accumulate cell culture related data related to cell culture, and to monitor and manage cell culture based on the cell culture related data. It is intended to provide a cell culture system, a cell culture method, and a cell culture program.
  • a cell culture device includes a culture container for culturing cells, medium storage means for storing a medium supplied to the culture container, and an image of the cells in the culture container.
  • An image acquisition means for acquiring, and a control means for determining a culture state of the cell from an image of the cell acquired by the image acquisition means and executing a culture operation based on the determination. It is.
  • the culture container according to the invention of claim 1 is characterized in that the culture container has a growth culture container for growing cells and a function of the cells.
  • a culture container for function expression for causing expression, and a fraction of cells in the culture container for function expression.
  • the image acquisition means acquires an image.
  • the cell culture device is the invention according to claim 2, wherein the culture container for function expression is for stimulating an inducer that stimulates cells with an inducer for proliferation.
  • the control means determines the cell growth possibility and the cell growth ability based on the image of the cells in the induction factor stimulation culture vessel, and controls the growth factor from the induction factor stimulation culture vessel. It is characterized by controlling the culture operation such as the timing of transferring the cells to the culture container and the supply of the culture medium from the culture medium storage means to the culture container for proliferation.
  • the cell culture device is the invention according to claim 2, wherein the functional expression culture vessel is a differentiation-inducing culture vessel for differentiating cells and is controlled.
  • the means is characterized in that the differentiation induction operation is controlled based on the image of the cells in the differentiation induction culture vessel.
  • the medium storage means is configured in a cassette structure and connected to a culture vessel. It is characterized by.
  • the cell culture device according to the invention of claim 6 is the invention according to claim 5, wherein the culture container for function expression is configured in a cassette structure, and the culture container for proliferation and the medium reservoir It is characterized by being connected to the means.
  • the cell culture device according to the invention of claim 7 is the invention according to any one of claims 2 to 6, wherein the culture medium storage means, the culture container for function expression and the culture container for proliferation are in a closed system. It is characterized by being configured.
  • the cell culture device according to the invention of claim 8 is the cell culture device according to any of claims 2 to 7, wherein the growth culture container is provided with a liquid reservoir so as to be formed. In the initial stage of culture in the culture vessel, cells and culture medium are stored in the liquid reservoir.
  • the cell culture device according to the invention of claim 9 is the culture according to any one of claims 2 to 8, wherein the cells grown in the culture vessel for proliferation are cultured for functional expression.
  • the cell image is guided into the container and the image of the cell is acquired by the image acquisition means. is there.
  • the cell culture device is the invention according to any one of claims 2 to 9, wherein the medium storage means stores a used medium storage capable of storing a used medium.
  • the container is installed together with a medium storage container capable of storing the medium to be supplied to the culture container, and the used medium in the growth culture container is discharged and stored in the used medium storage container. It is a feature.
  • the culture medium storage means can be equipped with a cell recovery container for recovering cells.
  • the cells concentrated in the culture vessel for proliferation are configured to be collected in the cell collection vessel.
  • a cell culture device according to the invention of claim 12 is characterized in that, in the invention of any one of claims 1 to 11, the cell is a suspension cell. is there.
  • the cell culture device according to the invention of claim 13 is characterized in that, in the invention of any one of claims 1 to 12, the cells are used in immunocell therapy. It is a thing.
  • the image acquisition means acquires an image of the cells in the culture vessel in which the cells are cultured, and from the cell image acquired by the image acquisition means.
  • the culture state of the cell is determined, and based on this determination, the culture operation is performed on the culture vessel.
  • the culture container according to the invention of claim 14 is characterized in that the culture container has a growth culture container for proliferating the cells and a function of the cells.
  • a culture container for function expression for expression, and an image acquisition means acquires an image of cells in the culture container for function expression.
  • the cell culture method according to the invention of claim 16 is the induction of the invention of claim 15, wherein the culture container for function expression stimulates cells with an inducer for proliferation.
  • This is a factor stimulation culture vessel, and based on the image of the cells in the induction factor stimulation culture vessel, the cell growth possibility and the cell growth ability are determined, and the above-described induction factor stimulation culture vessel is used for the growth culture. Timing of cell transfer to the container, medium storage means, etc. A culture operation such as supply of a culture medium to a culture vessel for breeding is performed.
  • the culture container for function expression is a culture container for differentiation induction for differentiating cells.
  • the differentiation induction operation is performed based on the image of the cells in the differentiation-inducing culture vessel.
  • the cell culture method according to the invention of claim 18 is the invention according to any one of claims 15 to 17, in the initial stage of culture in the culture container for proliferation, The cell and the culture medium are stored in the liquid reservoir.
  • the cell culture method according to the invention of claim 19 is the functional culture according to the invention of any one of claims 15 to 18, wherein the cells grown in the growth culture vessel are cultured.
  • the cell is guided into the container, and the image acquisition means acquires the cell image.
  • the cell culture method according to the invention of claim 20 is the invention according to any one of claims 15 to 19, wherein the spent medium in the growth culture container is used as the spent medium of the medium storage means. It is characterized by discharging into a storage container.
  • the cell culture method according to the invention of claim 21 is the cell culture method according to any one of claims 15 to 20, wherein the cells concentrated in the culture vessel for proliferation are recovered by the cell storage means. It collects in a container, It is characterized by the above-mentioned.
  • the cell culture method according to the invention of claim 22 is characterized in that, in the invention of any of claims 14 to 21, the cell is a suspension cell. .
  • the cell culture method according to the invention of claim 23 is characterized in that, in the invention of any one of claims 14 to 22, the cell is used in immune cell therapy. It is.
  • the cell culture program according to the invention of claim 24 is a cell culture program stored in a computer for executing cell culture, and acquires an image of a cell in a culture vessel in which the cell is cultured.
  • a cell culture program according to the invention of claim 25 is a cell culture program stored in a computer for executing cell culture, and a culture container for function expression for causing a cell to express a function.
  • the cell culture device according to the invention of claim 26 is the cell culture device according to any one of claims 1 to 3, wherein the culture vessel is placed on a mounting table. The culture area is changed by raising and lowering a part of the pedestal.
  • the cell culture device according to the invention of claim 27 is a simple fed-batch culture and a perfusion culture according to the invention according to claim 1 of claim 1 to claim 13. One of these can be selected.
  • the cell culture device according to the invention of claim 28 is characterized in that, in the invention of claim 27, one of intermittent perfusion culture and continuous perfusion culture can be selected. Is.
  • the cell culture method according to the invention of claim 29 is the invention according to any one of claims 14 to 16, wherein the culture container is placed on a mounting table, The culture area is changed by moving a part of the table up and down.
  • the cell culture method according to the invention of claim 30 is a simple fed-batch culture and a perfusion culture according to the invention of claim 14 to claim 23, wherein: One of these can be selected.
  • the cell culture method according to the invention of claim 31 is characterized in that, in the invention of claim 30, one of intermittent perfusion culture and continuous perfusion culture can be selected. Is.
  • the cell culture system according to the invention of claim 32 is a cell culture system for culturing cells and monitoring and managing the culture, each of which is provided with a culture vessel for culturing the cells.
  • the management means is installed in a culture chamber together with a thermostat, and in each culture unit of the thermostat An operation control panel having a function of controlling culture, and a monitoring computer that is installed outside the culture room and receives and displays data of the operation control panel.
  • the cell culture-related data includes cells, a medium, a culture vessel, a thermostat, a culture unit, and an operator. At least one of the identification code, the culture environment data in the thermostat and the culture unit, and the image data of the cells in the culture vessel.
  • a remote monitoring computer is connected to the monitoring computer via a public communication line. It is characterized by that.
  • the cell culture system according to the invention of claim 37 is the invention according to any of claims 32 to 36, wherein each of the canisters of the thermostatic chamber constituting the culture unit is a bacterium or the like. It is characterized by being isolated in a state where there is no entry / exit.
  • the cell culture system according to the invention of claim 38 is the invention according to any one of claims 32 to 37, wherein the canister of the thermostatic chamber constituting the culture unit includes A fan is installed to guide the air to the canister, and all the canister fans in one temperature chamber are stopped when the temperature chamber door is opened. It is structured.
  • the cell culture system according to the invention of claim 39 is the invention as set forth in any of claims 32 to 38, wherein the canisters of the thermostat bath constituting the culture unit are each canister in one thermostat bath.
  • the door force is V, and only one of the gaps is opened.
  • the cell culture system according to the invention of claim 40 is characterized in that, in the invention of any of claims 32 to 39, the cell is a suspension cell.
  • the cell culture system according to the invention of claim 41 is characterized in that, in the invention according to any one of claims 32 to 40, the cell is used in immune cell therapy.
  • a cell culturing method according to the invention of claim 42 is a cell culturing method for culturing cells and monitoring and managing the culturing, wherein a plurality of culturing containers for culturing the cells are provided.
  • Cell culture-related data related to cell culture for each culture unit in the thermostat using a thermostat that cultivates cells in an independent culture environment for each culture unit.
  • the cell culture state is collected for each culture unit based on the cell culture related data, and the cell culture operation is managed.
  • the cell culture-related data in the invention of claim 42 includes cells, a medium, a culture vessel, a thermostat, a culture unit, and an operation. It is at least one of the identification code of the person, the culture environment data in the thermostatic chamber and the culture unit, and the image data of the cells in the culture vessel.
  • the cell culture method according to the invention of claim 44 is characterized in that, in the invention of any one of claims 42 or 43, the cell is a suspension cell.
  • the cell culture method according to the invention of claim 45 is characterized in that, in the invention according to any one of claims 42 to 44, the cell is used in immune cell therapy. .
  • the cell culture program according to the invention of claim 46 is a cell culture program stored in a computer for culturing cells and monitoring and managing the culture, and each of the culture containers for culturing the cells. Multiple culture units are placed in isolation from each other. A procedure for collecting and accumulating cell culture-related data related to cell culture for each culture unit of the thermostat for culturing cells in an independent culture environment for each culture unit, and the cell culture-related data. And a procedure for monitoring the cell culture state for each culture unit, and a procedure for managing the cell culture operation for each culture unit based on the cell culture-related data.
  • a plurality of culture containers for culturing cells are sequentially connected, and each culture container cultures cells in a different culture environment, and the above-described culture on the downstream side
  • a feature is that the cultured cells are transferred to a container and cultured.
  • the cell culture device according to the invention of claim 48 is the culture according to the invention of claim 47, wherein the two culture vessels are installed, and one culture vessel expresses the function of the cells. It is a culture container for function expression having an environment, and the other culture container is a culture container for proliferation having a culture environment for growing cells.
  • the culture container for function expression has a culture environment in which a cell is stimulated by an inducing factor for proliferation. It is characterized by being a culture vessel for stimulating induction factors.
  • the cell culture device according to the invention of claim 50 is the differentiation-inducing culture container according to the invention of claim 48, wherein the culture container for function expression has a culture environment for differentiating the proliferated cells. It is characterized by being.
  • a cell culture device according to the invention of claim 51 is characterized in that, in the invention of any one of claims 47 to 50, the cell is a suspension cell.
  • a cell culture device according to the invention of claim 52 is characterized in that, in the invention of any one of claims 47 to 51, the cell is used in immune cell therapy.
  • the cells are cultured in a plurality of culture vessels in different culture environments, and the cells cultured in one culture vessel are placed downstream. It is characterized in that it is transferred to another culture vessel and cultured.
  • the number of the culture containers is two, and an inducing factor is used for growth in one culture container. After the cells are stimulated, the cells are grown in another culture vessel.
  • the number of the culture containers is two, and after the cells are grown in one culture container, It is characterized by differentiating cells in another culture vessel.
  • the cell culture method according to the invention of claim 56 is characterized in that, in the invention of any of claims 53 to 55, the cell is a suspension cell.
  • the cell culture method according to the invention of claim 57 is characterized in that, in the invention of any of claims 53 to 56, the cell is used in immune cell therapy.
  • a cell culture device is a cell culture device comprising a culture container for culturing cells and a mounting table for mounting the culture container, wherein the mounting table Has an ascending / descending portion, and the cultivatable area of the placed culture vessel changes as the ascending / descending portion moves up and down.
  • a cell culture device according to the invention of claim 59 is characterized in that, in the invention of claim 58, the cell is a suspension cell.
  • a cell culture device according to the invention of claim 60 is characterized in that, in the invention of claim 58 or claim 59, the cell is used for immune cell therapy.
  • a cell culture device according to the invention of claim 61 is the culture medium supplied to the culture container in the invention according to any one of claims 58 to 60.
  • a culture medium storage means for storing is connected.
  • the cell culture device according to the invention of claim 62 is characterized in that, in the invention of claim 61, the medium storage means and the culture vessel are configured in a closed system.
  • the culture medium storage means is a used culture medium storage container capable of storing a used culture medium. Is installed together with a medium storage container capable of storing the medium to be supplied to the culture container, and is configured to discharge and store the used medium in the growth culture container into the used medium storage container. To do.
  • the image of the cells in the culture container according to the invention according to any one of claims 58 to 63, V, shift 1 An image photographing means for obtaining the cell culture medium from the cell image obtained by the image obtaining means. And a control means for determining a feeding situation and executing a culture operation based on the determination.
  • the culture container is a growth culture container for growing cells, and causes the cells to exhibit functions.
  • an image acquisition means for acquiring an image of cells in the culture container for function expression.
  • the culture container for function expression stimulates a cell with an inducing factor for proliferation.
  • the control means determines the cell growth possibility and the cell growth ability based on the image of the cells in the induction factor stimulation culture container, and changes the culturable area.
  • Medium storage means force Controlling culture operations such as supply of culture medium to the growth culture vessel.
  • the culture state of the cell is determined from the image of the cell in the culture container, and the culture is performed. Since the culturing operation according to the situation is performed, the culturing state of the cell can be determined in a non-contact state, so that the cell is not damaged. Therefore, it is possible to reduce the labor of a person, and furthermore, it is possible to carry out a culture operation according to the state of cell culture, so that an appropriate culture operation can be realized. By realizing an appropriate culturing operation according to the culturing state of the cells, the culturing operation can be performed in hour units, and the culturing period can be shortened.
  • the medium storage means force S is configured in the S-cassette structure and connected to the culture container (the culture container for function expression, the culture container for proliferation), the culture Since the container can always be maintained in an optimum environment for culturing, damage to the cells in the culture container due to environmental changes can be reduced, and an aseptic operation for supplying the culture medium to the culture container in a clean bench or the like can be omitted.
  • the medium storage means, the culture container for function expression and the culture container for proliferation are configured in a closed system, It is possible to keep the medium storage means, the culture container for function expression and the culture container for proliferation in a sterile state.
  • the cells grown in the growth culture vessel are guided into the function expression culture vessel, and an image of the cells is acquired by the image acquisition means. Therefore, the cells grown in the growth culture vessel can be obtained as an image, and the number of cells and the morphology of the cells can be observed.
  • the cell collection container is subjected to centrifugal separation by recovering the entire amount of the cells concentrated in the growth culture container in the cell collection container of the medium storage means.
  • the cell can be collected by mounting it directly on the machine, and labor saving of the cell collection work can be realized.
  • the operation control panel and the monitoring computer are provided with a cell related to cell culture for each culture unit of the thermostat. Since culture-related data is collected and accumulated, it is possible to accurately grasp the culture history of cells cultured in any culture unit.
  • the operation control panel and monitoring computer are based on cell culture-related data for each culture unit of the thermostat. Since the cell culture state is monitored, the culture state abnormality can be monitored for each culture unit.
  • the management means is installed in the culture chamber together with the thermostat, and an operation control panel having a function of controlling the culture in each culture unit (caster and culture cassette) of the thermostat, and a monitoring room other than the above culture room Installed in the culture unit in the constant temperature bath in the culture room.
  • the culture state of the cells can be monitored, monitored and managed in a monitoring room other than the culture room using a monitoring computer. This management automatically records and stores the history etc. This prevents tampering and recording errors.
  • the canisters of the thermostatic chamber constituting the culture unit are each isolated by the intake filter and the exhaust filter so that cells or Z and bacteria do not enter and exit. Therefore, contamination in which cells in the culture bag and cell inoculation cassette stored in each caster are contaminated with bacteria can be prevented. Since the blower fans of all the casters in one temperature chamber are configured to stop when the main body door of the temperature chamber is opened, each of the temperature chambers in the temperature chamber is opened Since the canister can be kept sealed, an independent culture environment can be secured for each canister, and fluctuations in the culture environment within the canister can be suppressed.
  • one of the caster doors of each caster in one temperature chamber is configured to open, only one or more of the caster doors of each caster is opened simultaneously in one temperature chamber. Therefore, it is possible to prevent a culture bag in the canister from being mixed and a cell inoculation cassette to be carried in and out, and to prevent cross contamination where cells are contaminated with each other.
  • the initial stage of culture in the culture bag Antibody stimulation and cell growth in the same culture bag, and the liquid reservoir in which cells and culture medium are stored can be changed within a predetermined area, so that cells per area during culture can be changed.
  • the density at a density suitable for growth cells can be efficiently grown.
  • FIG. 1 (a) is a configuration diagram showing the first embodiment of the cell culture device according to the present invention
  • FIG. 1 (b) shows the first embodiment of the cell culture system according to the present invention
  • FIG. FIG. 2 is a perspective view showing the thermostat of FIG.
  • FIG. 5 is a layout diagram showing the configuration of one caster in the thermostatic chamber of FIG. 2, a culture cassette housed in the caster, and a powerful culture unit.
  • a cell culture device 10 shown in Fig. 1 (a) is a device that specifically cultures suspension cells used for immune cell therapy, and is equipped with a plurality of (for example, eight) culture units 12. 11, an operation control panel 13 for controlling the operation of the thermostat 11 and the culture unit 12, an image processing computer 14 for processing cell images, an operation control panel 13 and an image processing computer 14 And a monitoring computer 15 for monitoring the thermostatic chamber 11 and the culture unit 12.
  • the operation control panel 13 and the image processing computer 14 function as control means.
  • the cell culture device 10 can also be realized as a cell culture system.
  • the cell culture system 10 is a device that cultivates floating cells used for immune cell therapy, and monitors and manages the culture.
  • the thermostat 11, the operation control panel 13 and the image processing computer 14 are installed in a culture room (clean room) 94 suitable for cell culture, and the monitoring computer 15 is It is installed in a monitoring room 95 other than the culture room 94.
  • the thermostat 11 has a plurality of (for example, eight) culture units 12 arranged so as to be separated from each other, each of which is provided with a culture vessel for cell culture (a culture vessel for growth described later, an induction factor stimulation culture vessel). Is. this In the thermostat 11, the cells in the culture vessel are cultured in an independent culture environment for each culture unit 12.
  • a culture vessel for cell culture a culture vessel for growth described later, an induction factor stimulation culture vessel.
  • the operation control panel 13 controls the operation of the thermostat 11 and the culture unit 12, and monitors and manages cell culture for each culture unit 12 of the thermostat 11.
  • the image processing computer 14 processes an image of cells being cultured for each culture unit 12 in the thermostat 11, and functions as a control means for controlling the culture operation together with the operation control panel 13.
  • the monitoring computer 15 is connected to the operation control panel 13 and the image processing computer 14 to receive, store and display (view) data stored in the operation control panel 13 and the image processing computer 14. It can function as a management means for monitoring and managing cell culture together with the operation control panel 13.
  • the suspension cells peripheral blood mononuclear cells, LAK cells (Lymphokine Activated Killer cells), neural stem cells, ES cells and the like are known. These floating cells are hereinafter simply referred to as cells. These cells are cultured after being stimulated with an inducer suitable for each cell. Each inducer differs from cell to cell, for example, anti-human CD3 antibody in LAK cells, epithelial growth factor such as EGF in neural stem cell inducer, and fiber such as FGF-8b in inducer of ES cells It is a blast growth factor.
  • the cell culture device 10 can also be applied when culturing adhesion-dependent cells other than the suspension cells.
  • the culture unit 12 includes a plurality of (for example, eight) casters 16 (Fig. 2 and Fig. 5) separated from each other in the thermostatic chamber 11 and housed in each caster 16.
  • Culture cassette 17 (FIGS. 2 and 3).
  • the culture cassette 17 includes a culture bag 18 as a growth culture container, a cell inoculation cassette 19 as an induction factor stimulation culture container, and a medium cassette 20 as a medium storage means.
  • the cells in these culture vessels (culture no. 18, cell inoculation cassette 19) are cultured in an independent culture environment for each caster 16.
  • the thermostatic chamber 11 is provided with a multi-stage shelf 23 in a thermostatic chamber body 22 having a body door 21 that can be opened and closed, and one canister 16 is provided on each shelf 23. Be placed.
  • the thermostatic chamber 11 is closed to the environment (temperature, Maintain humidity and C02 concentration) in the environment necessary for culturing the cells.
  • a temperature sensor 24, a C02 sensor 25, a door sensor 26, and a heater 27 are disposed in the thermostatic chamber body 22.
  • Gas cylinder 28 installed outside is connected. Signals from the temperature sensor 24, the C02 sensor 25, and the door sensor 26 are transmitted to the operation control panel 13.
  • This operation control panel 13 controls the heater 27 based on the temperature signal from the temperature sensor 24, and controls the heater 27, and from the gas cylinder 28 to the thermostat body 22 based on the C02 concentration signal from the C02 sensor 25. Control the amount of C02 gas supplied to the inside.
  • the caster 16 has a caster door 31 attached to the caster body 30, and an intake filter 32 and an exhaust filter attached to the caster body 30. 33 is installed, and a blower fan 34 is installed on the intake filter 32 side of the caster body 30.
  • the intake filter 32 and the exhaust filter 33 are filters for removing bacteria, and when the air in the thermostat body 22 and the C02 gas are taken into the caster 16 by the operation of the blower fan 34, the thermostat tank Prevents inflow of bacteria from the body 22 into the caster 16.
  • the operation of the blower fan 34 is controlled by the operation control panel 13, and the operation of the blower fan 34 is performed when a signal indicating that the main body door 21 of the thermostatic chamber 11 is opened is transmitted from the door sensor 26 to the operation control panel 13. Is stopped, and the sealed state of the canister 16 is secured.
  • the function of preventing the inflow of bacteria from the thermostatic chamber body 22 into the caster 16 by the intake filter 32 and the exhaust filter 33, and ensuring the tightness of the caster 16 by stopping the operation of the blower fan 34, is provided for each carrier.
  • the inside of the star 16 is an independent culture environment.
  • the cells in the culture cassette 17 stored in one caster 16 in the thermostatic chamber 11 are isolated from the cells in the other casters 16 and stored in this caster 16. Contamination of cells in culture cassette 17 contaminated with various bacteria is prevented. Also, so-called cross-contamination that is contaminated by other patient's cells is prevented.
  • the caster body 30 is further provided with a door sensor 35, a door lock sensor 36, a temperature sensor 37, a humidity sensor 38, a door lock mechanism 39, a heater 40, and a circulation fan 41.
  • the operation control panel 13 controls the heater 40 based on the temperature signal from the temperature sensor 37. Ma
  • the operation control panel 13 controls the operation of the circulation fan 41 to circulate air and C02 gas in the canister 16.
  • the humidity sensor 38 detects the humidity in the canister 16 and transmits it to the operation control panel 13 to detect the abnormality. In this way, the inside of the canister 16 is maintained in an optimum environment for culturing cells.
  • the operation control panel 13 controls the operation of the door lock mechanism 39 so that two or more caster doors 31 do not open simultaneously in one thermostatic chamber 11. As a result, it is possible to prevent cells and culture media from being mixed and transferred between different casters 16.
  • the lock operation of the door lock mechanism 39 is detected by the door lock sensor 36 and transmitted to the operation control panel 13.
  • the open / close state of the canister door 31 is detected by the door sensor 35 and transmitted to the operation control panel 13.
  • a stage 42 is provided for supporting the culture cassette 17 stored in the caster 16, and a weighing scale 43 is installed on the stage 42.
  • This weighing scale 43 measures the weight of the culture bag 18 of the culture cassette 17 housed in the caster 16, and actually measures the amount of medium supplied from the culture cassette 20 to the culture bag 18. To do.
  • the measured value of the weigh scale 43 is also transmitted to the operation control panel 13.
  • the caster body 30 is provided with an indicator lamp 44. When automatic culture is performed in the caster 16, the indicator light 44 of the caster 16 is lit in red, for example, and the canister 16 for which an operation command is output and the culture cassette 17 is not housed inside.
  • the culture cassette 17 is a large tray 45 equipped with a culture bag 18, a cell inoculation cassette 19, and a medium cassette 20.
  • the culture bag 18 and the cell inoculation cassette 19 are A culture vessel for culturing cells.
  • the cell inoculation cassette 19 is a culture container for function expression for causing the cells to express functions (for example, proliferating the cells, differentiating the cells (described later), etc.).
  • Inducer for It is a culture vessel for stimulating an inducing factor that stimulates cells.
  • the culture bag 18 is a growth culture container for growing cells stimulated by the inducer in the cell inoculation cassette 19.
  • the culture bag 18 is a flexible disposable container for storing the culture solution inoculated with the cells.
  • the culture bag tray shown in Fig. 4 (A) is placed via the mounting table 46 (Fig. 5).
  • the culture bag tray 47 is detachably mounted on the large tray 45 shown in FIG. 3 and FIG.
  • the culture bag 18 is, for example, a nod which also has an oxygen-permeable material strength.
  • a first pump 48, a second pump 49, and a third pump 50 are arranged in the large tray 45.
  • One end side of the culture bag 18 is connected to the seventh connector 57 through the second pump 49 using the tube 63. Further, the other end of the culture bag 18 is connected to the first connector 51 using a tube 60.
  • the first pump 48, second pump 49, and third pump 50 are preferably peristaltic pumps because of the convenience of sterilization tube replacement.
  • one end of the tube 61 is connected to the third connector 53 and the other end is connected to the fifth connector 55.
  • the tube 61 is disposed in a rotary drive unit (not shown) of the first pump 48.
  • One end of the tube 63 is connected to the seventh connector 57 as described above, and the other end is connected to the culture bag 18.
  • the tube 63 is disposed in a rotation drive unit (not shown) of the second pump 49.
  • One end of the tube 62 is connected to the tube 61 by a connector X, and the other end is connected to the tube 63 by a connector Y.
  • the tube 62 is disposed in a non-illustrated rotary drive unit of the third pump 50.
  • the cell inoculation cassette 19 solidifies the inducer on the inside of the bottom surface of the inducer stimulating vessel 65, and puts the medium into the inducer stimulating vessel 65.
  • the medium is inoculated with cells, and this inducing factor stimulating vessel 65 is installed in the cassette frame 66, and is configured in a force set structure.
  • the operation of putting the inducer, medium and cells into the inducer stimulation container 65 is performed in a sterile condition in a clean bench or a safety cabinet (hereinafter referred to as a clean bench or the like in the present embodiment).
  • the mixed solution of the medium and the cells is referred to as a culture solution.
  • the cell inoculation cassette 19 is detachably attached to the large tray 45 as shown in FIG. Is done.
  • the second connector 52 and the fourth connector 54 of the cell inoculation cassette 19 are aseptically coupled to the first connector 51 and the third connector 53 of the large tray 45, respectively. That is, the first connector 51 and the second connector 52 are aseptically coupled by inserting the other needle-like coupling portion into one rubber-like coupling portion, for example. The same applies to the connection between the third connector 53 and the fourth connector 54.
  • the cell inoculation cassette 19 and the culture bag 18 in different culture environments, that is, the cells are stimulated with an inducing factor, and the cells are proliferated.
  • the cell inoculation cassette 19 having a culture environment for expressing the cells and the culture bag 18 having the culture environment for growing cells are connected. Therefore, the cells that have been stimulated by the inducer in the cell inoculation cassette 19 and have started to grow can be transferred to the culture bag 18, and only the growth can be performed in this culture bag 18.
  • the medium cassette 20 has a medium bag 67 as a medium storage container and a used medium bag 68 as a used medium storage container on a medium bag tray 69. Placed into a cassette structure.
  • the culture medium bag 67 stores the culture medium supplied to the culture knob 18 and the cell inoculation cassette 19.
  • the used medium bag 68 stores the used medium (supernatant) discharged from the culture bag 18. Since the culture cassette 20 is configured in the cassette structure, the culture bag 18 is maintained in the canister 16 and the culture cassette 17 in the canister 16 is simply installed in the culture cassette 17 to replace and supply the culture medium. Is possible.
  • the medium cassette 20 is detachably attached to the large tray 45 (Fig. 3).
  • the sixth connector 56 and the eighth connector 58 of the medium cassette 20 are connected to the fifth connector 55 and the seventh connector 57 of the large tray 45, respectively, and the first connector 51 and the second connector 52.
  • the culture medium bag 67 and the cell inoculation cassette 19 are connected.
  • the spent medium bag 68 and the culture bag 18 are connected by coupling the seventh connector 57 and the eighth connector 58.
  • the medium force in the medium bag 67 in the medium cassette 20 is activated by the activation of the first pump 48.
  • a closed system is constructed in which the cells are supplied to the culture bag 18 through the cell inoculation cassette 19 and the used medium in the culture bag 18 is discharged to the used medium bag 68 of the medium cassette 20 when the second pump 49 is activated. The With this closed system configuration, the system (culture bag 18, cell inoculation cassette 19 and medium cassette 20 is maintained in a sterile state.
  • the cell inoculation cassette 19 is replaced with a dummy cassette 70 when there are no more cells inside. This prevents the inducer in the cell inoculation cassette 19 from moving into the culture bag 18. Further, the medium cassette 20 is replaced with a new medium cassette 20 including a medium bag 67 filled with a medium and an empty used medium bag 68 when the medium bag 67 is empty. This exchange is performed by the operator. The dummy cassette 70 simply functions as a flow path for flowing the culture medium.
  • the first pump 48 is activated and the medium in the medium bag 67 of the medium cassette 20 is supplied (fed) to the culture bag 18.
  • the fed-batch culture in which the cells are propagated with the first pump 48 and the second pump 49 are started, and the spent medium in the culture bag 18 is discharged to the spent medium bag 68 of the medium cassette 20 and the medium no.
  • perfusion culture in which cells are grown by supplying the culture medium in the bag 67 to the culture bag 18, shaking culture using a shaking device 80 described later, and stationary culture without shaking.
  • perfusion culture includes intermittent perfusion culture in which spent medium discharge and medium supply are alternately performed, and continuous perfusion culture in which spent medium discharge and medium supply are performed simultaneously.
  • a filter 71 that normally prevents cell migration is disposed between the culture bag 18 and the second pump 49 in the tube 63, and the cells in the culture bag 18 are used as spent medium.
  • the bag 68 is prevented from being discharged.
  • the third pump 50 is activated when, for example, observing cells grown in the culture bag 18 with images. That is, if the filter 71 is not provided in the tube 63, the cells in the culture bag 18 are also transferred to the cell inoculation cassette 19 or the dummy cassette 70 through the tube 63, the tube 62, and the tube 61. The number of the proliferated cells is observed by being guided and imaged by a CCD force sensor 88 described later.
  • the filter 71 is disposed in the tube 63
  • the upstream side of the third pump 50 is connected to the culture bag 18 via the bypass tube 73, and the cells in the culture bag 18 are transferred to the bypass tube. Then, the light is introduced into the cell inoculation cassette 19 or the dummy cassette 70 through the tube 73 and the tube 62, and observed using the CCD camera 88.
  • the spent medium in the culture bag 18 is discharged into the used medium bag 68 of the medium cassette 20 and the cells in the culture bag 18 are concentrated. .
  • the spent medium is discharged by starting the second pump 49, and the volume of the culture solution in the culture bag 18 is reduced to about 1Z2 to about LZ3 by the control of the operation control panel 13 based on the measured value of the weighing scale 43. It is carried out until. Concentration of the cells in the culture bag 18 reduces the number of times of centrifugation by a centrifuge for the purpose of subsequent cell washing and concentration.
  • the used medium bag 68 is replaced with a cell collection bag 72 as a cell collection container that can be attached to a centrifuge.
  • the culture solution (medium and cells) in which the cells are concentrated in the culture bag 18 may be supplied into the cell collection bag 72 by starting the second pump 49. In this case, it is a condition that the tube 71 is not provided with the filter 71.
  • the cells can be collected in the canister 16 that is a closed system space by using a bag that can be attached to the centrifuge, and the work of collecting the cells can be saved.
  • the cell inoculation cassette 19 or the dummy cassette 70 is attached to the large tray 45.
  • a cell inoculation cassette sensor 74 to detect and a medium cassette sensor 75 to detect that the medium cassette 20 is mounted on the large tray 45 are installed.
  • the signals of these sensors 74 and 75 are transmitted to the operation control panel 13.
  • the operation control panel 13 confirms that the cell inoculation cassette 19 or the dummy cassette 70 is mounted on the large tray 45 of the culture cassette 17 and the medium cassette 20 is mounted, and then the first pump 48 and the second pump 49. And start the third pump 50.
  • the culture cassette 17 is housed in the caster 16 and the force by which the culture cassette 17 is supported by the stage 42 of the caster 16
  • a tilt motor 76, a cam mechanism 79, and a positioning sensor 77 are installed at a lower position where the culture bag tray 47 of the culture cassette 17 is arranged.
  • Culture bags on culture bag tray 47 18 The mounting table 46 that directly mounts a part thereof (not shown) is configured so that it can be moved up and down.
  • the tilt motor 76 rotates the cam mechanism 79 to raise and lower the elevating part of the mounting table 46.
  • the position of the lift is detected by the positioning sensor 77 and transmitted to the operation control panel 13.
  • the tilt motor 76 is controlled by the operation control panel 13 and is controlled to lower the elevating part of the mounting table 46 at the initial stage of culture in the culture bag 18. As a result, a liquid reservoir 78 (FIGS. 8 and 9) is formed in the culture nodule 18.
  • the culture solution (medium and cells) from the cell inoculation cassette 19 is stored in the liquid reservoir 78, thereby increasing the cell density per area in the culture bag 18. Therefore, the cells are efficiently proliferated at the initial stage of culture.
  • the tilting motor 76 raises the elevating part of the mounting table 46 via the cam mechanism 79 to keep the culture bag 18 in a horizontal state. As a result, the liquid reservoir 78 is eliminated.
  • a shaking mechanism 91 of a shaking device 80 is installed as a pressing means at an upper position where the culture bag tray 47 of the culture cassette 17 is arranged.
  • the shaking device 80 includes the shaking mechanism 91, the operating motor 81, the cam mechanism 90, and the position determining sensor 82.
  • an operation plate 85 is disposed on the apparatus frame 83 via a guide rod 84 so as to be movable up and down, and a plurality of protrusions 86 protrude from the bottom surface of the operation plate 85. It was established.
  • the actuating plate 85 is alternately moved upward or downward by the actuating motor 81 by the action of the cam mechanism 90 (Fig.
  • the culture bag 18 is repeatedly pressed, that is, the culture bag 18 is repeatedly pressed and released. As a result, the culture solution in the culture bag 18 is agitated, the cells in the culture bag 18 float and move in the culture solution, and the distribution of cells and the concentration of the components in the culture bag 18 are reduced. Cell growth is promoted by homogenization and increased oxygen supply capacity.
  • the position of the operation plate 85 is detected by the positioning sensor 82 and transmitted to the operation control panel 13, and the operation motor 81 is controlled by the operation control panel 13.
  • the cell culture (shaking culture) in the culture bag 18 using the shaking device 80 may be performed before the culture solution in the culture bag 18 is filled to about 1 or 2 times. It may be implemented after being done.
  • an LED 87 for illumination is obtained above the position where the cell inoculation cassette 19 or the dummy cassette 70 of the culture cassette 17 is arranged, and an image is obtained below.
  • a CCD camera 88 is installed as a means.
  • the illumination LED 87 illuminates the cell inoculation cassette 19 or the dummy cassette 70 from above.
  • the CCD camera 88 captures the image of the cells in the cell seed cassette 19 or the dummy cassette 70 with a downward force, and acquires the image.
  • the illumination operation of the LED 87 for illumination and the photographing operation of the CCD camera 88 are controlled by the operation control panel 13 (Fig. 6), and the cells in the cell inoculation cassette 19 or the dummy cassette 70 are controlled every predetermined time (for example, 6 hours).
  • An image is acquired.
  • the cell image at every predetermined time is stored in the image memory circuit 89 of the image processing computer 14.
  • the image processing computer 14 performs image processing, for example, binarization processing or multi-value processing, on a cell image for each predetermined time stored in the image memory circuit 89, thereby obtaining a single cell projection area. And the rate of increase of non-single cells, which are cell aggregates in which single cells aggregate, are calculated as evaluation parameters for cell culture.
  • the average value of the projected area of a single cell was determined by mounting the cell inoculation cassette 19 on the culture cassette 17 and storing the culture force set 17 in the canister 16 to start the culture.
  • the time force is also calculated from the cell image after 24 hours, for example.
  • a force is a non-single cell as described above is based on the fact that the projected area of a single cell at the initial stage of culture is less than 100 ⁇ m 2 , and therefore the non-single case where the projected area is 100 m 2 or more.
  • the rate of increase of non-single cells is calculated by calculating the change in the ratio of non-single cells to total cells from images of cells over time (for example, images after 24, 48, and 72 hours from the start of culture). Is calculated.
  • the image processing computer 14 also calculates the lag time for the average projected area force of a single cell, and estimates the growth start time of the cell.
  • the lag time is the time required for the induction period from inoculation of cells into the inducer stimulating vessel 65 of the cell inoculation cassette 19 until the start of growth.
  • the image processing computer 14 determines the culture status of the cell from the start of cell proliferation, that is, whether or not the cell has a possibility of proliferation by stimulation with an inducer. Then, the image processing computer 14 determines the cell determination result (for example, the cell If there is a possibility that the cells will grow, “YES”, if not, a signal such as “NO”) is transmitted to the operation control panel 13.
  • the operation control panel 13 When the operation control panel 13 receives a signal indicating that the possibility of growth of the cells in the induction factor stimulating container 65 of the cell inoculation cassette 19 is extremely low, the operation control panel 13 displays the state of the cells. It should be noted that cells with too long lag time are cells that are not easily stimulated by an inducing factor, and are judged to have a low possibility of proliferation.
  • the image processing computer 14 calculates the minimum doubling time of the cell from the increasing rate of the non-single cell.
  • the double time is the time required for the number of cells in a certain time to double.
  • the image processing computer 14 determines the culture state of the cell, that is, the proliferation ability of the cell from the minimum doubling time, and transmits it to the operation control panel 13.
  • the operation control panel 13 that receives the signal from the image processing computer 14 then moves the cells from the cell inoculation cassette 19 to the culture bag 18 based on the above-described cell growth ability, and transfers the medium to the culture bag 18. Determine the flow acceleration to be fed. It should be noted that a cell with a minimum fold time that is too long is determined to be a cell with extremely low proliferation ability.
  • the operation control panel 13 and the image processing computer 14 functioning as control means are a CPU that executes computation and control, a storage device (memory) that stores processing programs and data, and data. And an input device such as a keyboard for inputting commands and commands, an input device such as a mouse or touch panel, and an output device such as a monitor. Further, the image processing computer 14 includes an image memory circuit 89 for storing image data of the CCD camera 88 force.
  • images of cells in the cell inoculation cassette 19 taken every predetermined time by the CCD camera 88 are subjected to image processing (for example, binarization processing or multi-value processing).
  • image processing for example, binarization processing or multi-value processing.
  • Cell culture evaluation parameters average projected area of single cell, non-single cell increase acceleration), etc., and cell culture status (cell growth possibility, cell growth) from this cell culture evaluation parameter A program for determining the ability
  • the storage device of the operation control panel 13 includes devices related to the thermostat 11, the carriage 16 and the culture cassette 17 according to the cell culture conditions (for example, the first pump 48, the second pump 49, etc.) Is stored, and a program for executing the culturing operation is stored. Further, the storage device of the operation control panel 13 is based on signals from various sensors of the thermostat 11, the canister 16, and the culture cassette 17, such as controlling the CCD camera 88 every predetermined time to acquire cell images. A device control program for controlling devices related to the thermostatic chamber 11, the canister 16, and the culture cassette 17 is also stored.
  • the recording device of the operation control panel 13 is provided for each caster 16 of the thermostat 11 and for each of these.
  • a program for monitoring the cell culture state and a program for managing the cell culture operation for each of the caster 16 and the culture cassette 17 based on the cell culture-related data are stored.
  • the cell culture-related data are as follows: cells, medium, inducer, culture bag 18, large tray 45, medium bag 67, medium cassette 20, inducer stimulation container 65, cell inoculation cassette 19, cell recovery Back 72, operator's ID, thermostat 11, and each address of caster 16, etc.
  • Identification code that can be read with a barcode reader, thermostat 11, caster 16 and culture cassette, for example 17 sensors (temperature sensor 24, door sensor 35, medium cassette sensor 75, weigh scale 43, etc.) or detected by various devices (first pump 48, second pump 49, blower fan 34, operating motor 81, etc.)
  • Culture environment data indicating the operation state, cell image data acquired by the image processing computer 14 (image data by the CCD camera 88, image data after binarizing the image, and the like) At least one of the evaluation parameters, etc.) that issued calculated from the data.
  • the operation control panel 13 acquires, collects and accumulates cell culture-related data at predetermined times (for example, every minute) for each of the canister 16 and the culture cassette 17 in the thermostatic chamber 11 based on the above program. To do. Thereby, the culture history regarding the cells cultured in the arbitrary canister 16 and the culture cassette 17 in the thermostat 11 is automatically acquired regardless of the operator. Similarly, the operation control panel 13 observes the culture state of the cells based on the culture-related data for each of the caster 16 and the culture cassette 17 in the thermostat 11 and monitors whether there is an abnormality.
  • the operation control panel 13 replaces the culture medium cassette 20 for each of the caster 16 and the culture cassette 17 of the thermostatic chamber 11 (medium replacement operation), the replacement operation to the dummy cassette 70, and the cell recovery bag 72.
  • manage various culture operations such as the replacement operation, and notify the operator, etc. that the operation is encouraged. With this management, history and other information can be automatically recorded and stored, preventing artificial tampering and recording errors.
  • the monitoring computer 15 includes cell culture-related data, culture history data, data on presence / absence of abnormality, data on culture operation for each of the canister 16 and the culture cassette 17 of the thermostat 11 included in the operation control panel 13. Is received from the operation control panel 13 and saved so that it can be displayed (viewed) on the monitor. As a result, the operator observes and monitors the culture of the cells cultured in the thermostatic chamber 11 in the monitoring chamber 95 other than the culture chamber 94 where the thermostatic bath 11 is installed, and manages and manages it. It becomes possible to do. With this management, history and other information can be recorded and stored automatically, preventing human falsification and recording errors.
  • a bar code reader is used to inoculate the inducer stimulating vessel.
  • 65 IDs, IDs assigned to the storage containers for the induction factor and medium, cell samples, and operator IDs are acquired by the operation control panel 13 together with the operation date and time and transmitted to the monitoring computer 15.
  • the inducer stimulation container 65 is mounted on the cell inoculation cassette 19
  • the barcode reader is used with the ID of the induction factor stimulation container 65, the ID of the cell inoculation cassette 19 and the operator ID together with the operation date and time. Is acquired by the operation control panel 13 and transmitted to the monitoring computer 15.
  • the ID of the culture bag 18, the ID of the large tray 45, and the operator ID are acquired by the operation control panel 13 using the bar code reader together with the operation date and time. Sent to the monitoring computer 15.
  • the culture medium bag 67 and the used culture medium bag 68 are mounted on the culture medium cassette 20, the ID of the culture medium back 67, the ID of the culture medium cassette 20 and the operator ID are used together with the operation date and time, and a no code reader is used. Is acquired by the operation control panel 13 and transmitted to the monitoring computer 15.
  • the addresses of the thermostat 11 and the caster 16, the ID of the large tray 45, and the operator is acquired together with the operation date and time by the operation control panel 13 using a bar code reader and transmitted to the monitoring computer 15.
  • the medium cassette 20 is mounted on the large tray 45 stored in the canister 16 of the thermostat 11, the addresses of the thermostat 11 and the canister 16, the ID of the medium cassette 20, and the operator ID are stored.
  • the operation date and time it is acquired by the operation control panel 13 using a bar code reader and transmitted to the monitoring computer 15.
  • the addresses of the thermostat 11 and canister 16 and the cell inoculation cassette 19 are acquired together with the operation date and time by the operation control panel 13 using a barcode reader and transmitted to the monitoring computer 15.
  • the canister Operation and stop status of various devices such as the blower fan 34 and the first pump 48
  • measurement data of various sensors temperature sensor 37, weight scale 43, etc.
  • the image data of cells taken by the CCD camera 88, the processing data obtained by binarizing the image data, and evaluation parameters, along with the date and time and the address of the caster 16, are processed. Obtained from the computer 14 to the operation control panel 13 and transmitted to the monitoring computer 15. At this time, the operation control panel 13 outputs control signals for controlling various devices to the thermostat 11, the caster 16 and the culture cassette 17 as described above.
  • the lock mechanism 39 is activated to release the door lock, and the signal from the cell inoculation cassette sensor 74 or the medium cassette sensor 75 is replaced with the above dummy force set 70, the medium cassette 20 is replaced (medium replacement), the cell Confirm replacement with collection bag 72.
  • Fig. 20 when cells are collected in the cell collection bag 72 after cell culture is completed, the corresponding constant temperature is removed when the medium cassette 20 equipped with the cell collection bag 72 is removed.
  • Address of tank 11 and caster 16, ID of medium cassette 20, ID of cell recovery bag 72 The operator ID is acquired by the operation control panel 13 together with the operation date and time, and is transmitted to the monitoring computer 15. Thereafter, when the cell collection bag 72 is removed from the medium cassette 20 with a clean bench or the like, the ID of the medium cassette 20, the ID of the cell collection bag 72 and the operator ID are acquired in the operation control panel 13 together with the operation date and time. Sent to monitoring computer 15
  • the operation control panel 13 and the image processing computer 14 execute the above-described program to culture the cells.
  • the operation control panel 13 and the image processing converter 14 set and control the culture environment independently for each canister 16 in the thermostatic chamber 11, and the culture cassettes stored in each canister 16. Cells in 17 are cultured.
  • FIG. 8 to FIG. 13 show the induction factor-stimulated intermittent perfusion culture process, in which cells are collected in the culture bag 18.
  • the operator places an empty culture bag 18 on the culture bag tray 47 via the mounting table 46, and places the culture bag tray 47 on the large tray 45.
  • the culture bag 18 is connected to the first pump 48, the second pump 49, and the third pump 50.
  • the operator carries the large tray 45 into the single canister 16 of the thermostatic chamber 11 in which the indicator lamp 44 is lit in green, for example, and supports it on the stage 42. Then, the operator solidifies the inducing factor in the inducing factor stimulating vessel 65 in a clean bench or the like, puts the medium, and inoculates the cells with the cell inoculation cassette 19, as shown in FIG. Mount on the large tray 45 in the caster 16. Subsequently, the operator attaches the medium cassette 20 in which the medium is put into the medium bag 67 in the clean bench or the like to the large tray 45 in the caster 16 (Sl in FIG. 12).
  • the operator outputs an image output from the CCD camera 8 8 in the caster 16 in which the culture tray 18 having the culture bag 18, the cell inoculation cassette 19 and the medium force set 20 mounted on the large tray 45. Confirm (S2 in Fig. 12). Before or after confirming the image output, the operator activates the tilt motor 76 of the canister 16 to lower the elevating part of the mounting table 46 and form the liquid reservoir 78 in the culture bag 18. Further, the operator measures the weight of the empty culture bag 18 with the weight meter 43 of the caster 16. [0144] After that, as shown in FIG. 9A, the operator closes the caster door 31 and starts culturing cells in the canister 16 (S3 in FIG. 12).
  • the cells are stimulated by the inducer for growth in the inducer stimulation container 65 of the cell inoculation cassette 19 (S4 in FIG. 12).
  • the CCD camera 88 of the caster 16 images the cells in the induction factor stimulation container 65 of the cell inoculation cassette 19 every predetermined time (for example, 6 hours), and the image processing computer 14 uses this imaging image power. Cell culture evaluation parameters are calculated. Further, the image processing computer 14 also calculates the lag time for this evaluation parameter force, determines whether the cell is stimulated by the induction factor and has a possibility of proliferation, and further calculates the minimum doubling time. And determine the proliferation ability of the cell (S5 in FIG. 12).
  • the operation control panel 13 receives the growth from the image processing computer 14 even if the cells are stimulated by the inducer in the inducer stimulation container 65 of the cell inoculation cassette 19 and the force is also applied for a predetermined time (eg, 72 hours). When a signal is received that the possibility is not recognized, a message to that effect is displayed. When the operation control panel 13 receives a signal from the image processing computer 14 that the cells in the induction factor stimulating vessel 65 of the cell inoculation cassette 19 have been determined to be proliferating, the operation control panel 13 Based on the above, the timing for transferring the cells to the culture bag 18 and the flow acceleration of the medium into the culture bag 18 are determined.
  • a predetermined time eg, 72 hours
  • the operation control panel 13 operates the first pump 48 to feed the medium in the medium bag 67 of the medium force set 20 to the cell inoculation cassette 19 as shown in FIG.
  • the cells in the inducer stimulation container 65 of the cell inoculation cassette 19 are transferred to the culture bag 18 and the medium in the medium bag 67 is fed to the culture bag 18 (S6 in FIG. 12).
  • the operation control panel 13 controls the culture solution in the culture bag 18 measured by the weighing scale 43. It is determined whether or not the weight exceeds the predetermined value b (S10 in FIG. 12). When the weight exceeds the predetermined value b, the operation motor 81 is started. As a result, the shaking device 80 operates as shown in FIG. 10 (B), and shaking culture in which the operating plate 85 in the shaking mechanism 91 of the shaking device 80 repeatedly presses the culture bag 18 is started (FIG. 12). Sl l). The operation control panel 13 continues to determine whether or not the weight of the culture solution in the culture bag 18 measured by the weigh scale 43 has reached the predetermined value c (S12 in FIG. 12).
  • the first pump 48 is stopped, the feeding of the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18 is stopped, the operation motor 81 is stopped, and the inside of the culture bag 18 is stopped. Is stopped (S 13 in FIG. 13).
  • the operation control panel 13 operates the second pump 49, and as shown in Fig. 11 (A), the spent medium (culture medium) in the culture bag 18 is operated. The supernatant in the bag 18) is discharged into the used medium bag 68 of the medium cassette 20 (S14 in FIG. 13). Thereafter, the operation control panel 13 activates the first pump 48 to feed the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18, starts the operation motor 81, and uses the shaking device 80 to incubate the culture bag 18. The shaking culture is performed inside (S15 in FIG. 13).
  • the operation control panel 13 stops the first pump 48 to stop the feeding of the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18, stops the operating motor 81, and the culture bag 18 The shaking culture in the inside is stopped (S16 in Fig. 13).
  • the operation control panel 13 determines whether or not the force has reached a desired culture period depending on the use date and time of the cells to be proliferated, or the image processing computer 14 force cells in the culture bag 18 Is determined whether or not the power reaches the desired number of cells (S17 in FIG. 13).
  • the operation control panel 13 activates the third pump 50 (Fig. 11 (A)), and the cells in the culture bag 18 are activated. A part of the cell is transferred to the cell inoculation cassette 19 or the dummy cassette 70 (in most cases, the dummy cassette 70), and the CCD camera 88 images the cells.
  • the image of the cell is processed by the image processing computer 14, and it is determined whether the number of cells is equal to or greater than a specified value and transmitted to the operation control panel 13.
  • the operation control panel 13 repeats the processing operations of steps S14 to S17 when the culturing period or the number of cells has not been reached.
  • steps S13 to S17 are performed by discharging the culture medium used in the culture bag 18 and Is an intermittent perfusion culture in which new medium supply (feeding) is performed alternately in
  • steps S9 to S17 the presence or absence of cells in the induction factor stimulation container 65 of the cell inoculation cassette 19 is confirmed by the CCD camera 88, and the operation control panel 13 confirms that the cells are in the induction factor stimulation container 65. If it does not exist, the operator is prompted to replace the cell inoculation cassette 19 with the dummy cassette 70 so that the induction factor does not flow into the culture bag 18 from the induction factor stimulation vessel 65. When replacing the dummy cassette 70, the operation control panel 13 completes the replacement of the medium cassette 20 into the culture bag 18 from the medium cassette 20 and the shaking culture by the shaking device 80. Suspend until
  • Steps S9 to S17 when the operation control panel 13 determines that the medium has disappeared in the medium bag 67 of the medium power set 20 from the measured value of the weight scale 43, the medium cassette 20 is newly replaced. Encourage the operator to replace the media cassette 20. Even when the medium cassette 20 is replaced, the operation control panel 13 can replace the medium cassette 20 with the medium fed from the medium cassette 20 to the culture bag 18 and the shaking culture with the shaking device 80. Suspend until complete.
  • the operation control panel 13 When the operation control panel 13 reaches the desired culture period or reaches the desired cell number in step S17, the operation control panel 13 stops shaking, and after cell sedimentation, as shown in Fig. 11 (B).
  • the second pump 49 is activated, the spent medium in the culture bag 18 is discharged to the spent medium bag 68 of the medium cassette 20, and the culture solution in the culture bag 18 is discharged based on the measured value of the weighing scale 43.
  • About 1Z2 ⁇ Concentrate cells to about LZ3 (S18 in Fig. 12).
  • the operation control panel 13 then stops the second pump 49 and ends the cell culture (S 19 in FIG. 12). After completion of the culture, the operator moves the cells in the culture bag 18 to a centrifuge container in a clean bench or the like, and then collects the cells by centrifugation (S20 in FIG. 12).
  • FIGS. 14 and 15 show a case where the same factor-stimulated intermittent perfusion culture process includes a process of collecting cells with the cell collection bag 72 (FIG. 5). Accordingly, steps S21 to S38 in the steps shown in FIGS. 14 and 15 are the same as steps S1 to S18 in FIGS. [0156]
  • step S38 shown in Fig. 15 after the cells in the culture bag 18 are concentrated by starting the second pump 49, the operation control panel 13 stops the second pump 49 and the medium cassette 20 has been used. The operator is prompted to replace the medium bag 68 with the cell collection bag 72 (FIG. 5) (S39 in FIG. 13).
  • the cell collection bag 72 is a bag that can be attached to a centrifuge and used for centrifugation.
  • the operation control panel 13 activates the second pump 49 and the operating motor 81, and the shaking bag 80 causes the culture bag 18 to be activated. While shaking the inside, the cells in the culture bag 18 are transferred together with the medium to the cell recovery bag 72 attached to the medium cassette 20 (S40 in FIG. 15). The operation control panel 13 then stops the second pump 49 and the operating motor 81 to stop the cell culture (S41 in FIG. 15). After completion of the culture, the operator attaches the cell collection bag 72 to the centrifuge and collects the cells by centrifugation (S42 in FIG. 15).
  • Steps S51 to S62 in the induction factor stimulation continuous perfusion culture process shown in FIGS. 16 and 17 are the same as steps S1 to S12 in the induction factor stimulation intermittent perfusion culture process of FIGS. Is omitted.
  • a filter 71 is disposed between the culture bag 18 and the second pump 49.
  • the operation control panel 13 is fed with the medium from the medium bag 67 of the medium cassette 20 into the culture bag 18 (S56 in Fig. 16), and during the shaking culture by the shaking device 80 in the culture bag 18 ( (S61 in Fig. 16)
  • the second pump 49 is activated and the spent medium in the culture bag 18 is removed. Drain the media cassette 20 into the used media bag 68.
  • continuous perfusion culture in which feeding of the medium to the culture bag 18 and discharging of the spent medium from the culture bag 18 is started in the culture bag 18 (S63 in FIG. 17).
  • the cells in the culture bag 18 are blocked from flowing by the filter 71 and do not flow into the spent medium bag 68.
  • shaking culture with the shaking device 80 is also performed at the same time.
  • the operation control panel 13 reaches a desired culture period depending on the use date and time of the cells to be proliferated. Or whether or not the image processing computer 14 has reached the desired number of cells in the culture bag 18 (S64 in FIG. 17). When determining whether or not the cell has reached the desired number of cells in the culture bag 18, the operation control panel 13 activates the third pump 50 to transfer some of the cells in the culture bag 18 to the cell inoculation cassette. Move to 19 or dummy force set 70 (in most cases, dummy cassette 70) and have the CCD camera 88 take a picture of this cell.
  • the image of the cell is processed by the image processing computer 14, and it is determined whether or not the number of cells is equal to or greater than a specified value, and is transmitted to the operation control panel 13. Then, the operation control panel 13 repeats the continuous perfusion culture in step S63 when the culture period or the number of cells has not been reached.
  • steps S59 to S64 the presence or absence of cells in the induction factor stimulation container 65 of the cell inoculation cassette 19 is confirmed by the CCD camera 88, and the operation control panel 13 is installed in the induction factor stimulation container 65.
  • the operator is prompted to replace the cell inoculation cassette 19 with the dummy cassette 70 so that the induction factor does not flow into the culture bag 18 from the induction factor stimulation vessel 65.
  • the operation control panel 13 completes the replacement of the medium cassette 20 into the culture bag 18 from the medium cassette 20 and the shaking culture by the shaking device 80 into the dummy cassette 70. Suspend until
  • steps S59 to S64 when the operation control panel 13 determines that the medium is no longer in the medium bag 67 of the culture force set 20 from the measured value of the weight scale 43, the operation control panel 13 renews the medium cassette 20. Encourage the operator to replace the media cassette 20. Even when the medium cassette 20 is replaced, the operation control panel 13 can replace the medium cassette 20 with the medium fed from the medium cassette 20 to the culture bag 18 and the shaking culture with the shaking device 80. Suspend until complete.
  • the operation control panel 13 stops the first pump 48, the second pump 49, and the operation motor 81 at the time when the desired culture period in step S64 or the desired number of cells is reached, and the perfusion is performed.
  • the culture and shaking culture are stopped (S65 in FIG. 17).
  • the operation control panel 13 starts the second pump 49, discharges the used medium in the culture bag 18 to the used medium bag 68 of the medium cassette 20, and cultures based on the measured value of the weighing scale 43. Concentrate the cells until the culture in bag 18 is about 1Z2 to 1Z3. (S66 in Fig. 17).
  • the reason for stopping the shaking device during the concentration process is to prevent a large amount of cells from entering the tube and clogging the filter.
  • the operation control panel 13 then stops the second pump 49 and ends the cell culture (S 67 in FIG. 17). After this culture is completed, the operator moves the cells in the culture bag 18 to a centrifuge container in a clean bench or the like, and then collects the cells by centrifugation (S68 in FIG. 17).
  • the image of the cells in the cell inoculation cassette 19 imaged by the CCD camera 88 is processed by the image processing computer 14 to evaluate the cell culture evaluation parameters (average single cell projection area, non-single cell The rate of increase) is obtained, and the culture status (proliferation potential and proliferation ability of the cell) is evaluated.
  • the operation control panel 13 performs culture operations according to the culture conditions (such as the timing of cell transfer from the cell inoculation cassette 19 to the culture bag 18 and the addition of medium at a predetermined flow acceleration from the medium cassette 20 to the culture bag 18). ).
  • the culture conditions can be determined in a non-contact * non-invasive state, thus avoiding the risk of contamination and cell loss due to sampling that does not damage the cells.
  • the cells of one patient are inoculated into the cell inoculation cassette 19 of the culture cassette 17 housed in a single caster 16, and the culture operation according to the culture state of the cells is performed for each of these cells. Therefore, it is possible to realize an appropriate culture operation for each patient and to avoid cross contamination.
  • the culturing operation can be performed in units of time, culturing is promoted, and the culturing period can be shortened.
  • the medium cassette 20 is configured in a cassette structure and connected to the culture bag 18 and the cell inoculation cassette 19 also configured in a cassette structure, It can always be kept in the caster 16 in the optimum environment for culture.
  • the medium can be exchanged by simply opening and closing the main body door 21 of the thermostat 11 and the caster door 31 of the caster 16, it is possible to reduce the number of times the doors 21 and 31 are opened and closed. 11 and the change of the culture environment in the caster 16 can be suppressed. As a result, damage to the cells in the culture bag 18 due to environmental changes can be reduced, and aseptic operation for supplying the culture medium to the culture bag 18 in a clean bench or the like can be omitted.
  • the protrusion 86 of the working plate 85 in the shaking mechanism 91 of the shaking device 80 repeatedly presses the flexible culture bag 18 containing the medium inoculated with the cells, so that the inside of the culture bag 18 Since the culture solution is stirred, the cell distribution in the culture bag 18 and the concentration of the components in the medium are made uniform, and the oxygen supply ability is increased, thereby promoting cell growth. Cell culture efficiency can be improved.
  • the operation control panel 13 and the monitoring computer 15 collect and accumulate cell culture-related data related to cell culture for each culture unit 12 (canister 16 and culture cassette 17) in the thermostatic chamber 11. Therefore, it is possible to accurately grasp the culture history of the cells cultured in any culture unit 12.
  • the operation control panel 13 and the monitoring computer 15 monitor the cell culture state for each culture unit 12 (caster 16 and culture cassette 17) in the thermostatic chamber 11 based on cell culture-related data.
  • the culture state abnormality can be monitored for each culture unit 12.
  • the operation control panel 13 and the monitoring computer 15 replace the medium (exchange medium cassette 20) for each culture unit 12 (caster 16 and culture cassette 17) in the thermostat 11 based on the cell culture-related data. ) And cell collection after culturing (cell collection with culture bag 18 or cell collection bag 72), etc., and operations performed by the operator for each culture unit 12 in the thermostat 11 (operation) A work schedule can be easily created.
  • the management means is installed in the culture chamber 94 together with the thermostat 11, and has a function of controlling the culture in each culture unit 12 (the caster 16 and the culture cassette 17) of the thermostat 11.
  • An operation control panel 13 and a monitoring computer 15 which is installed in a monitoring room 95 other than the culture room 94 and receives, stores and displays (views) data held by the operation control panel 13.
  • the state of cell culture in the culture unit 12 in the thermostat 11 in the culture chamber 94 is observed and monitored in a monitoring room 95 other than the culture room 94 using the monitoring computer 15 and managed. can do.
  • history and other information can be recorded and stored automatically, preventing human falsification and recording errors.
  • the casters 16 of the thermostatic chamber 11 constituting the culture unit 12 are configured to be separated from each other by the intake filter 32 and the exhaust filter 33 so that cells or Z and bacteria do not enter and exit. Therefore, culture bags 18 and cells stored in each caster 16 It is possible to prevent contamination of the cells in the inoculation cassette 19 with bacteria.
  • each caster 16 in one thermostat 11 Since only one of the caster doors 31 of each caster 16 in one thermostat 11 is open, each caster 16 in one thermostat 11 Since two or more of the caster doors 31 are not opened at the same time, it is possible to prevent the culture bag 18 and the cell inoculation cassette 19 from being transferred in and out of the caster 16, and the cells are contaminated with each other. Cross contamination can be prevented.
  • FIG. 21 is a layout diagram showing the configuration (differentiation induction state) of the culture unit in the second embodiment of the cell culture device according to the present invention.
  • FIG. 22 is a layout diagram showing the configuration of the culture unit of FIG. In the second embodiment, the same parts as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.
  • the cell culture device 100 according to the second embodiment is different from the cell inoculation cassette 19 (Fig. 5) in the cell culture device 10 according to the first embodiment in the differentiation induction culture in which a differentiation inducing factor is added in advance. It is replaced with a sorting guide cassette 101 as a container.
  • this cell culture device 100 after the medium is introduced from the medium bag 67 of the medium cassette 20 to the culture bag 18 by starting the second pump 49 to proliferate the cells, the proliferated cells are transferred to the first pump 48. Is activated to give the cell a role, that is, the cell is differentiated.
  • differentiation means giving a cell a role as a heart cell, a role as a liver cell, or the like.
  • a dummy cassette 102 is arranged instead of the differentiation induction cassette 101 as shown in FIG.
  • Used medium power in culture bag 18 First cassette 48 activates medium cassette 2 via dummy cassette 102 It is discharged into a used medium bag 68 of 0. Similar to the dummy cassette 70 (FIG. 5), the dummy cassette 102 simply functions as a flow path for allowing the medium to flow.
  • the CCD camera 88 takes images of cells that are differentiated in the differentiation-inducing cassette 101 at predetermined time intervals.
  • Program power for executing this photographing is stored in the recording device of the operation control panel 13.
  • the image processing computer 14 performs, for example, a binarization process or a multi-value process on the cell image, and acquires the cell state as an evaluation parameter.
  • a program for executing the acquisition of the evaluation parameter is stored in the recording device of the image processing computer 14.
  • the image processing computer 14 also determines whether or not the cell has been differentiated with respect to the time-dependent change of the cell morphology.
  • the operation control panel 13 that has received a signal from the image processing computer 14 controls the differentiation induction operation by the differentiation induction cassette 101.
  • the operation control panel 13 continues the guidance operation, and otherwise displays that fact. To do.
  • a program for executing the above-described differentiation determination is recorded in the recording device of the image processing computer 14, and is stored in the recording device of the operation control panel 13 in order to execute the separation guidance operation.
  • a program for monitoring the cell culture state and a program for managing the cell culture operation are stored. Based on the above program, the operation control panel 13 collects and accumulates cell culture-related data for each of the canister 16 and the culture cassette 17 in the thermostat 11, almost as in the case of the first embodiment. Monitors the presence or absence of abnormalities and manages culture operations such as replacement of the medium cassette 20 (medium replacement operation) and replacement of the dummy cassette 102 with the dispensing guide cassette 101.
  • the monitoring computer 15 includes cell culture related data, culture history data, data on presence / absence of abnormality for each of the caster 16 and the culture cassette 17 of the thermostatic chamber 11, Data concerning the culture operation is received from the operation control panel 13 and stored, and can be displayed (viewed) on the monitor.
  • the cell culture-related data is acquired in the same manner as in the first embodiment.
  • the case for the guidance cassette 101 is different. That is, before cell culture (see FIG. 18), when the differentiation-inducing factor 103 is added to the differentiation-inducing container 103 (FIG. 21) of the differentiation-inducing cassette 101 in a talin bench or the like, the ID of this differentiation-inducing container 103, Differentiation induction factor ID and operator ID power The operation date and time are acquired by the operation control panel 13 and transmitted to the monitoring computer 15.
  • the differentiation induction container 103 is mounted on the differentiation induction cassette 101, it is acquired by the operation control panel 13 together with the ID of the differentiation induction container 103, the ID of the separation induction cassette 101, and the operator ID force operation date and time. Is sent to the computer 15.
  • the operation control panel 13 When the dummy cassette 102 is removed and replaced with the distribution induction cassette 101, the addresses of the thermostat 11 and caster 16, the ID of the differentiation induction cassette 101, the large tray 45 The ID and the operator ID are acquired by the operation control panel 13 together with the operation date and time, and transmitted to the monitoring computer 15.
  • the operator adds a medium and cells to the culture bag 18 in a clean bench or the like (S71 in FIG. 23), and places the culture bag 18 on the culture bag tray 47 via the mounting table 46. Attach this culture bag tray 47 to the large tray 45 (S72 in FIG. 23). Then, the operator carries the large tray 45 into the single canister 16 of the thermostatic chamber 11 in which the indicator lamp 44 is lit in green, for example.
  • the operator attaches the medium cassette 20 to the large tray 45 in the canister 16 and attaches the dummy cassette 102 (FIG. 22) (S73 in FIG. 23). Then, the operator closes the caster door 31 of the canister 16 and starts stationary culture of the cells in the culture bag 18 (S74 in FIG. 23). In this stationary culture, when the culture medium is initially small in the culture bag 18, the operator activates the tilt motor 76 to lower the elevating part of the mounting table 46, and the culture bag 18 has a liquid storage part 78. (Fig. 8) is formed.
  • the operation control panel 13 activates the second pump 49 to feed the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18 (S75 in Fig. 23).
  • the operation control panel 13 also confirms whether or not the culture solution in the culture bag 18 has exceeded the predetermined value a by checking the measured value force of the weigh scale 43 (S76 in FIG. 23). When the value exceeds the specified value a
  • the tilt motor 76 is activated to raise the elevating part of the mounting table 46, and the culture bag 18 is set in a horizontal state to eliminate the liquid reservoir 78 (S77 in FIG. 23).
  • the operation control panel 13 determines whether or not the weight of the culture solution in the culture bag 18 measured by the weigh scale 43 is equal to or greater than the predetermined value b (S78 in FIG. 23). At this point, the operating motor 81 is activated, and the shaking device 80 is activated to start shaking culture (S79 in FIG. 23). The operation control panel 13 continues to determine whether or not the force of the culture solution in the culture bag 18 measured by the weigh scale 43 exceeds the predetermined value c (S80 in FIG. 23). At this point, the second pump 49 is stopped, the feeding of the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18 is stopped, the operation motor 81 is stopped, and the inside of the culture bag 18 is stopped. Is stopped (S81 in FIG. 23).
  • the operation control panel 13 operates the first pump 48 to remove the spent medium (the supernatant in the culture bag 18) from the culture cassette 2 It is discharged into the used medium bag 68 of 0 (S82 in FIG. 24). Thereafter, the operation control panel 13 activates the second pump 49, feeds the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18, activates the operating motor 81, and the shaking device 80 in the culture bag 18 Shake culture at (S83 in Fig. 24). After a predetermined time has elapsed, the operation control panel 13 stops the second pump 49, stops feeding the medium from the medium bag 67 of the medium cassette 20 to the culture bag 18, stops the operation motor 81, and stops the culture bag. Stop shaking culture in 18 (S84 in Fig. 24)
  • the operation control panel 13 determines whether or not the force has reached a desired culture period depending on the use date and time of the cells to be proliferated, or the image processing computer 14 force cells in the culture bag 18 Is determined whether or not the force has reached the desired number of cells (S85 in FIG. 24).
  • the operation control panel 13 activates the third pump 50 to transfer some of the cells in the culture bag 18 to the dummy cassette 102. And let the CCD camera 88 take pictures of these cells.
  • the image processing computer 14 performs image processing on the cell image, determines whether or not the number of cells is equal to or greater than a specified value, and transmits the image to the operation control panel 13.
  • the operation control panel 13 repeats the operations of steps S82 to S85 when the culturing period or the number of cells has not been reached.
  • the above steps S81 to S85 are intermittent perfusion culture in which discharge of the used medium in the culture bag 18 and supply of new medium (feeding) into the culture bag 18 are performed alternately. Is
  • Steps S77 to S85 if the operation control panel 13 determines that the medium is no longer in the medium bag 67 of the medium cassette 20 from the measured value of the weight scale 43, the medium cassette 20 is updated. Encourage the operator to replace the sputum medium force set. Even when the medium cassette 20 is replaced, the operation control panel 13 replaces the medium cassette 20 with the addition of the medium from the medium cassette 20 to the culture bag 18 and the shaking culture with the shaking device 80. Suspend until is completed.
  • the operation control panel 13 activates the first pump 48 when the desired culture period is reached or the desired number of cells is reached in step S85, and the spent medium in the culture bag 18 is used for the medium cassette 20.
  • the cells are discharged into the finished medium bag 68, and the cells are concentrated until the culture solution in the culture bag 18 becomes about 1Z2 to 1Z3 based on the measurement value of the weight scale 43 (S86 in FIG. 24).
  • the operation control panel 13 activates the second pump 49 to feed the medium in the medium bag 67 of the medium cassette 20 to the culture bag 18, and to operate.
  • the motor 81 is activated and the culture bag 18 is shake-cultured with the shaking device 80 (S88 in FIG. 24).
  • the operation control panel 13 activates the first pump 48 and continues the culture in the culture bag 18 while shaking culture in the culture bag 18 and the flow of the medium from the culture cassette 20. Is moved to the distribution guide cassette 101 (S89 in FIG. 24).
  • sorting induction culture is started in the differentiation-inducing cassette 101 (S90 in FIG. 24).
  • the operation control panel 13 captures the cells in the sorting induction cassette 101 with the CCD camera 88 every predetermined time (for example, 6 hours).
  • the image processing computer 14 performs image processing on the captured image, acquires the cell morphology as an evaluation parameter, and determines whether or not the cell has been differentiated in the differentiation-inducing cassette 101 from the time-dependent change in the cell morphology. (S91 in Fig. 24).
  • the operation control panel 13 stops the second pump 49, the first pump 48, and the operating motor 81 when receiving the signal that the image processing computer 14 has also determined that the cells have been separated in step S91. Then, the differentiation-inducing culture is stopped and the cell culture is terminated (S92 in FIG. 24). After the completion of the culture, the operator collects the cells in the differentiation-inducing cassette 101 by a cell collection operation in a clean bench or the like. (S93 in Figure 24).
  • the cell culture apparatuses 10 and 100 both have cells cultured in one culture container among a plurality of culture containers for culturing cells in different culture environments, and transferred to another culture container on the downstream side. Yes.
  • cells are stimulated with an inducing factor for growth in the cell inoculation cassette 19 which is one culture container, and then the cell inoculation cassette 19 is cultured in another culture container.
  • the cells are transferred to the bag 18 to grow the cells.
  • the cell culture apparatus 100 after the cells are grown in the culture bag 18 which is one culture container, the cells are transferred from the culture bag 18 to the separation guide cassette 101 which is another culture container. The cells are separated.
  • cell culture equipment According to devices 10 and 100, variations in cell culture form can be expanded.
  • the image processing computer 14 performs image processing on the image of the CCD camera 88 to calculate an evaluation parameter, and based on the evaluation parameter, the cell culture status (cell proliferation possibility, cell proliferation The power described for determining the capability)
  • the operation control panel 13 may perform the function of the image processing computer 14.
  • cell images may be taken with the CCD camera 88 in the culture bag 18.
  • a filter 71 is placed between the second pump 49 and the culture bag 18. You don't have to.
  • the culture solution in order to grow cells, is stirred using the shaking device 80.
  • the culture bag 18 is vibrated up and down or left and right.
  • the medium may be swung to flow the culture solution to make the cell distribution and the medium component concentration uniform, and to increase the oxygen supply capacity.
  • the operation control panel 13 and the image processing computer 14 control each canister in one thermostat. However, there are a plurality of similar thermostats. The operation control panel 13 and the image processing computer 14 may execute control for each of the canisters in the thermostatic chamber.
  • the operation control panel 13 and the image processing computer 14 control the operation of each canister 16 in one thermostat 11, respectively.
  • the operation control panel 13 and the monitoring computer 15 monitor and manage the cell culture, a plurality of thermostats 11 as shown in the third embodiment shown in FIG.
  • One operation control panel 13 is arranged in the thermostat 11, and one image processing computer 14 and one monitoring computer 15 are connected to each operation control panel 13, respectively. It may be possible to observe, monitor, and manage the cell culture performed in each of the casters 16 of the plurality of thermostats 11. With this management, The history etc. can be recorded and stored automatically, preventing artificial tampering and recording mistakes.
  • the remote monitoring computer is connected to one monitoring computer 15 using the public communication line 104. 1 05 may be connected.
  • the remote monitoring computer is connected to one monitoring computer 15 using the public communication line 104. 1 05 may be connected.
  • a culture abnormality or the like in a specific canister 16 of a specific thermostat 11 can be quickly grasped at a remote place using the remote monitoring computer 105.
  • FIG. 26 is a configuration diagram showing a fourth embodiment of the cell culture device according to the present invention.
  • FIG. 27 shows one spatially independent culture chamber 240 and low temperature chamber 230 in the cell culture device 200 according to the fourth embodiment, and the culture bag tray 241 and the low greenhouse 230 stored in the culture chamber 240.
  • FIG. 6 is a layout diagram showing the configuration of a culture unit 212 composed of a medium bag tray 231 and a waste solution bag tray 232 stored in the container.
  • the cell culture device 200 shown in FIG. 26 is a device that specifically cultures floating cells used for immune cell therapy, and includes a culture device 210 having a plurality of (eg, three) culture units 212. All of the data, including the operation control PLC (programmable 'logic' controller) 223 for controlling the operation of the plurality of culture units 212, the image processing unit 221 for processing cell images, Data collection device 224 to be collected, touch panel 222 for displaying and inputting control information of operation control PLC 223 and image processing unit 221, operation control PLC 223, image processing unit 221, data collection device 224 via hub 225 And a monitoring computer 226 for monitoring the cell culture device 200 and the culture unit 212.
  • the operation control PLC 223 and the image processing unit 221 function as control means.
  • Each of the plurality of culture units 212 is equipped with an observation camera (CCD camera) 302.
  • peripheral blood mononuclear cells LAK cells (Lymphokine Activated Killer cells), neural stem cells, ES cells and the like are known as the suspension cells.
  • LAK cells Lymphokine Activated Killer cells
  • neural stem cells ES cells and the like are known as the suspension cells.
  • ES cells ES cells and the like are known as the suspension cells.
  • These floating cells are hereinafter simply referred to as cells.
  • This cell culture device 200 can also be applied when culturing adhesion-dependent cells (for example, mesenchymal stem cells) other than the above suspension cells.
  • the cell culture device 200 is a plurality of (for example, three) cultures that are spatially and structurally independent. In the structure in which the units 212 are stacked, each culture unit 212 is separated into a low temperature chamber 230 and a culture chamber 240 as shown in FIG.
  • a culture bag tray 241 In the cell culture apparatus 200, a culture bag tray 241, a medium bag tray 231 and a waste liquid bag tray 232 are stored.
  • a culture bag 242 as a culture container for antibody stimulation and proliferation is placed on the culture bag tray 241
  • a culture bag as a culture medium storage means is placed on the culture medium bag tray 231 and the waste solution bag tray 232.
  • (New medium bag) 233 and waste liquid nog (waste medium bag) 234 as a waste liquid storage means are mounted.
  • the culture unit 212 includes doors (not shown) that can be opened and closed in the culture chamber 240 and the low temperature chamber 230.
  • the culture unit 212 keeps the environment (temperature and C02 concentration) in the culture chamber 240 in an environment necessary for culturing cells while keeping the door closed, and the environment (temperature) in the low temperature chamber 230 is maintained. Maintain an optimal environment for medium storage.
  • the culture unit 212 is provided with temperature sensors 236 and 243, a C02 sensor 244, a door sensor (not shown), and a heater (not shown). Further, a gas cylinder (not shown) installed outside is connected to the cell culture apparatus 200 (culture unit 212) via a C02 supply system 245. Signals from the temperature sensors 236, 243, the C02 sensor 244 and the door sensor are transmitted to the operation control PLC 223.
  • This operation control PLC 223 controls the heater based on the temperature signal from the temperature sensors 236 and 243, and based on the C02 concentration signal from the C02 sensor 244, adjusts the gas cylinder power and the amount of C02 gas supplied into each culture unit 212. Control. C02 in the room is discharged by a predetermined amount by a circulating pump 246.
  • the operation of the stirring fan 247 is controlled by the PLC 223 for operation control, and the operation of the stirring fan 247 stops when a signal indicating that the door of the culture unit 212 is opened is also sent to the PLC 223 for operation control.
  • the environmental change in the culture unit 212 is alleviated.
  • the cell culture device 200 has a dry heat sterilization function, and the sterilization in the device and each tray can be sterilized with the culture bag tray, the medium bag tray, and the waste solution bag tray set. Yes, bacterial growth in the culture unit 212 can be prevented.
  • each culture unit 212 has an independent spatial structure, it is isolated from the cells in other culture units 212 and stored in the culture unit 212.
  • the culture bag 242, the medium bag 233, and the waste solution bag 234 are installed in the culture unit 212 after being connected in a clean bench or the like, so the culture bag 242 stored in the culture unit 212 is a closed system (not open) System), and contamination in which cells in the culture bag 242 are contaminated by various bacteria is prevented.
  • the culture unit 212 further includes a door sensor (not shown), a door lock sensor (not shown), a temperature sensor 236, 243, a door lock mechanism (not shown), a heater (not shown), and a stirring fan 247.
  • the operation control PLC 223 controls the heater based on the temperature signals from the temperature sensors 236 and 243.
  • the operation control PLC 223 controls the operation of the stirring fan 247 to circulate air and C02 gas in the culture chamber 240. In this way, the inside of the culture chamber 240 is maintained in an optimum environment for culturing cells.
  • the operation control PLC 223 controls the operation of the door lock mechanism so that the doors of two or more culture units 212 are not simultaneously opened in one cell culture device 200. As a result, it is possible to prevent cells and culture media from being mistakenly carried in between different culture units 212.
  • the lock operation of the door lock mechanism is detected from the door lock sensor and transmitted to the operation control PLC 223. Further, the open / closed state of the door of the culture unit 212 is detected by a door sensor and transmitted to the operation control PLC 223.
  • a frame 250 is provided to support the culture bag tray 241 stored in the culture chamber 240.
  • the weight of the frame 250 installed on the upper part of the culture unit 212 A total of 251 are installed in support.
  • This weigh scale 251 measures the weight of the culture bag 242 of the culture bag tray 241 housed in the culture chamber 240. In actuality, the amount of medium supplied from the culture medium bag 233 to the culture bag 242 is measured. And the amount of waste liquid discharged from the culture bag 242 to the waste liquid bag 234 is measured. The measured value of the weigh scale 251 is also transmitted to the operation control PLC 223.
  • the culture unit 212 is provided with an indicator lamp (not shown) that displays the presence / absence of the culture bag 242 in the culture unit 212.
  • the operation control PLC 223 lights the indicator light, for example, red, and the culture bag 242 is stored in the culture chamber 240! Sometimes, the indicator light is lit in green, for example.
  • the culture bag tray 241 will be described. As shown in FIG. 27, the culture bag tray 241 is equipped with a culture bag 242.
  • the culture bag 242 is a culture container for culturing cells.
  • the culture bag 242 is a culture container for function expression for expressing functions (eg, proliferating cells, differentiating cells, etc.) in the cells.
  • the cells are stimulated with antibodies for proliferation. It is a culture vessel for antibody stimulation. It is also a growth culture container for growing cells stimulated with antibodies in the same culture bag 242.
  • the culture bag 242 is a flexible container that stores a medium inoculated with cells, and is placed on the culture bag tray 241 via the placement table 252.
  • the culture bag 242 is, for example, a bag having an oxygen-permeable material strength.
  • a supply pump 261 and a discharge pump 271 are arranged in the culture chamber 240.
  • One end side of the culture bag 242 is connected to the supply system joint 264 through the supply pump 261 using the tube 262. Further, the other end side of the culture bag 242 is connected to a discharge system joint 274 via a discharge pump 271 using a tube 272.
  • the culture bag 242 is connected to the supply system joint 264 by the tube 262 and connected to the medium bag 233 by the tube 265.
  • the culture bag 242 is connected to the discharge system joint 274 via the tube 272 and also connected to the waste liquid bag 234 via the tube 275.
  • the antibody is solidified inside a part of the bottom surface connected to the supply pump 261 of the culture bag 242, and the culture bag 242 is filled with the medium.
  • Cells are inoculated into the culture medium, and the culture bag 242 is placed on the culture bag tray 241 to form a cassette structure.
  • the operation of solidifying the antibody in the culture bag 242 and placing the medium and cells is performed in a sterile condition in a clean bench or the like.
  • the medium bag tray 231 and the waste liquid bag tray 232 each have a medium bag 233 as a medium storage container and a waste liquid bag 234 as a used medium storage container, and are configured in a cassette structure. .
  • the culture medium bag 233 stores the culture medium supplied to the culture bag 242.
  • the waste liquid bag 234 stores a used medium (supernatant) from which the culture bag 242 is also discharged.
  • Medium bag tray 231 force With the S-cassette structure, the medium bag tray 231 is simply installed in the low temperature chamber 230 of the culture unit 212 while the culture bag 242 is maintained in the culture chamber 240. Can be exchanged and supplied.
  • the culture bag tray 241 is detachably attached to the culture unit 212.
  • the supply system joint 264 of the culture bag 242 is aseptically coupled to the joint of the culture medium bag 233
  • the discharge system joint 274 is aseptically coupled to the joint of the waste liquid bag 234, respectively. That is, the supply system joint 264 and the joint of the culture medium bag 233 are aseptically coupled by inserting the other needle-like coupling part into one rubber-like coupling part, for example.
  • the operation of combining the culture bag 242 with the culture medium bag 233 and the waste liquid bag 234 is performed in a sterile condition in a clean bench or the like.
  • the culture bag 242, the culture medium bag 233, and the waste liquid bag 234 are connected as described above, whereby the culture medium in the culture medium bag 233 in the culture medium bag tray 231 is supplied to the culture bag 242 when the supply pump 261 is activated,
  • a closed loop is configured in which the spent medium in the culture bag 242 is discharged to the waste solution bag 234 of the waste solution bag tray 232 when the discharge pump 271 is activated. With this closed loop configuration, the system (culture bag 242, culture bag 233, waste solution bag 234) is maintained in a sterile state.
  • the supply pump 261 is activated to supply the culture medium in the culture bag 233 of the culture medium bag tray 231 to the culture bag 242 (flow calorie). And the supply pump 261 and the discharge pump 271 are activated to discharge the spent medium in the culture bag 242 to the waste bag 234 in the waste bag tray 232 and the medium bag 233.
  • perfusion culture includes intermittent perfusion culture in which used medium is discharged and medium is supplied alternately, and continuous perfusion culture in which used medium is discharged and medium is supplied simultaneously.
  • a filter that prevents the movement of cells is usually disposed in the tube 272 between the culture bag 242 and the discharge pump 271, and the cells in the culture bag 242 are discharged to the waste liquid bag 234. It is prevented.
  • the spent medium in the culture bag 242 is discharged to the waste solution bag 234 of the waste bag tray 232, and the cells in the culture bag 242 are concentrated. .
  • the discharge of the spent medium is performed by starting the discharge pump 271. Based on the measurement value of the weighing scale 251, the volume of the medium and cells in the culture bag 242 is reduced to about 1Z2 to 1Z3 by the control of the operation control PLC223. It is carried out until.
  • the concentration of the cells in the culture bag 242 reduces the number of times of centrifugation by the centrifuge performed thereafter.
  • the waste bag tray 232 after the cells in the culture bag 242 are concentrated as described above, the waste bag 234 is replaced with a cell collection bag as a cell collection container that can be attached to a centrifuge.
  • the culture solution (medium and cells) in which the cells are concentrated in the culture bag 242 may be supplied into the cell collection bag by starting the discharge pump 271. In this case, it is a condition that the tube 272 is not provided with a filter.
  • the cells can be collected in a bag that can be attached to the centrifuge in the culture unit 212, which is a closed system space, and the work of collecting the cells can be saved.
  • a culture bag tray 241 is accommodated in the culture unit 212, and the force with which the culture bag tray 241 is supported by the frame 250 of the culture chamber 240 is on the culture nog tray 241.
  • the mounting table 252 on which the culture bag 242 is directly mounted is composed of a pad 252a that does not move up and down and a plurality of connected area change parts 252b and 252c that can be moved up and down. These area change parts 252b and 252c can control the culture area of the culture bag 242.
  • an inclination motor (elevating mechanism) 280, a cam mechanism 281 and a positioning sensor are installed at positions below the area changing parts 252b and 252c of the culture bag tray 241.
  • the tilt motor 280 rotates the cam mechanism 281 to raise and lower the area changing parts 252b and 252c of the mounting table 252 independently of each other.
  • the position of the elevator is detected by the positioning sensor and transmitted to the operation control PLC 223.
  • the tilt motor 280 is controlled by the operation control PLC 223, and is controlled to lower the area changing parts 252b and 252c of the mounting table 252 in the initial stage of culture in the culture bag 242.
  • a liquid reservoir is formed in a portion corresponding to the area changing parts 252b and 252c of the culture bag 242.
  • the mounting table 252 shown in FIG. 28 can change the culture area in three stages by raising and lowering the height of the area changing parts 252b and 252c. Three or more area-changing parts may be provided. This allows fine adjustment of the culture area.
  • FIG. 28 is a perspective view showing the configuration of the mounting table 252 of FIG.
  • the culture conditions suitable for antibody stimulation that is, the cell density in the culture bag 242 is maintained at a density suitable for growth.
  • the tilting motor 280 raises the area changing parts 252b and 252c of the mounting table 252 via the cam mechanism 281 to remove the culture bag 242.
  • the area of the liquid reservoir is changed by setting only a predetermined area of the park to a horizontal state.
  • the cell density per unit area in the culture bag 242 is maintained at a density suitable for growth by changing the area of the cells and the reservoir of the medium. It is propagated efficiently.
  • the mounting table 252 can form two liquid pools, first, the medium and cells are held in the lowest part of the area change part 252c in FIG. Avoid lowering.
  • the height of the area change part 252c in Fig. 28 is increased by one step, and the area change part 252b and the area change part 252c can be cultivated at the same height. Increase the area of the appropriate culture bag 242 by a certain amount. As a result, a cell density suitable for proliferation can be maintained for a certain period of time.
  • the shaking mechanism 291 of the shaking device 290 is installed as a pressing means at an upper position where the culture bag 242 of the culture bag tray 241 is arranged.
  • the shaking device 290 includes the shaking mechanism 291, the operating motor 292, the cam mechanism 293, and a positioning sensor (not shown). As shown in FIG.
  • an operating plate 291 a as a pressing means is movably arranged on the apparatus frame 250 via a guide rod 250 a, and a plurality of shaking plates 291 a are arranged on the bottom surface of the operating plate 291 a.
  • the protruding portion 291b is projected.
  • the operation plate 291a is alternately moved upward or downward by the operation of the cam mechanism 293 by the operation motor 292, so that the protrusion 291b of the operation plate 291a moves the culture bag 242 positioned below the shaking mechanism 291.
  • Repeated pressing, that is, pressing and releasing the culture bag 242 is repeated.
  • the medium in the culture bag 242 is agitated, the cells in the culture bag 242 float and move in the medium, and the cell distribution and oxygen concentration distribution in the culture bag 242 are made uniform, Cell proliferation is promoted.
  • FIG. 29 is a plan view showing the configuration of the mounting table 352 according to the modification of the fourth embodiment.
  • (A) is a part 353 that does not move up and down and all the area change parts 354, 355, 356,
  • FIG. 35 is a diagram showing a state where 357 is on the same plane
  • FIG. Fig. 30 is a partial cross-sectional view along the ⁇ - ⁇ line of Fig. 29 (a), where (a) shows the part 353, the area changing part 355, and the area not moving up and down by lowering the area changing part.
  • B shows the state where three of the four area change parts 355, 356, 357 are on the same plane.
  • C is a diagram showing a state in which the part 353 that does not move up and down and all the area change parts 354, 355, 356, and 357 forces S are on the same plane.
  • the mounting table 352 has an area changing part 357, 356, 355, 3 54 in the shape of an isosceles triangle having the corner as a vertex from one corner 352a of the rectangular in plan view. And parts 353 that do not move up and down. Parts 353 that do not move up and down are placed on a horizontal plane. As shown in Fig. 30, the part 353 and the area change part 354 that do not move up and down are connected by the connecting member 353a, and the area change part 354 and the area change part 355 are connected by the connecting member 354a.
  • the area changing part 355 and the area changing part 356 are connected to each other by a connecting member 355a, and the area changing part 356 and the area changing part 357 are connected to each other by a connecting member 356a.
  • a connecting member 355a As the connecting rods 353a, 354a, 355a, 356a, hinges can be used if they are arranged.
  • the number of area changing parts may be two to three, which is four, or may be five or more.
  • Support legs 365 and 367 are bonded and fixed to the lower surfaces of the area change parts 355 and 357, respectively.
  • the lower surface of the support leg 365 is a plane 365a and a plane 365b on the horizontal plane where the corner 352a side force is also arranged in order, and an inclined plane connecting these planes 365a and 365b.
  • the plane 365a is located below the plane 365b.
  • the lower surface of the support leg 367 includes the flat surfaces 367a, 367b, and 367c on the horizontal plane where the one side 352a side force of the mounting table 352 is also arranged in order, the inclined surface 367d that connects the flat surfaces 367a and 367b, and the flat surface 367b and the flat surface 367c.
  • An inclined surface 367e that connects the two, and also a force, the plane 367a is positioned below the plane 367b, and the plane 367b is positioned below the plane 367c.
  • the distance from the area change part 355 of the plane 365a is the same as the distance from the area change part 357 of the plane 367a, and the distance from the area change part 355 of the plane 365b is the same as the distance from the area change part 357 of the 367b. It is the same as the distance.
  • a height adjustment plate 370 that can be moved by the motor 380 along the ⁇ - ⁇ line in Fig. 29 (a) (in the direction of arrow A in Fig. 30 (b)). Arranged. On the height adjusting plate 370, elevating members 375 and 377 for elevating the area changing parts are bonded and fixed.
  • the upper surface of the elevating member 375 includes a plane 375b and a plane 375a on the horizontal plane in which the corner 352a side force of the mounting table 352 is also arranged in order, and these planes 375a and 375b.
  • the plane 375a is located above the plane 375b.
  • the upper surface of the elevating member 377 includes horizontal planes 377c, 377b, 377a, an inclined plane 377d that connects the plane 377a and the plane 377b, and a plane 377b and a plane.
  • the inclined surface 377e connecting the 377c and the force, the plane 377a is located above the plane 377b, and the plane 377b is located above the plane 377c.
  • Lifting member 377 supports the upper surface It has a shape corresponding to the lower surface of the leg 367, and when the inclined surfaces 377d, 377e are brought into contact with the inclined surfaces 367e, 367d of the support leg 367, the planes 377a, 377b, 377c are the planes 367c of the support leg 36 7 , 367b and 367a, respectively.
  • the distance from the top surface of the height adjustment plate 370 on the plane 375a is the same as the distance from the top surface of the height adjustment plate 370 on the plane 377a, and the distance from the top surface of the height adjustment plate 370 on the plane 375a. Is the same as the distance from the upper surface of the height adjustment plate 370 on the plane 377b.
  • the support leg 367 rises along the shape of the upper surface of the elevating member 377 ( (B)) o Thereby, the planes 367a and 367b of the support leg 367 and the inclined surface 367d force abut on the planes 377b and 377a and the inclined surface 377d of the elevating rod 377, respectively.
  • the elevating member 375 moves relative to the support leg 365 while the flat surfaces 365a and 365b and the flat surfaces 375b and 375a are in contact with each other until the inclined surfaces 365c and the inclined surfaces 375c are in force contact.
  • the three area changing parts 355, 356, and 357 out of the four area changing parts are in the same plane.
  • the support leg 367 further moves along the shape of the upper surface of the elevating member 377. It rises (Fig. 30 (c)). As a result, the flat surface 367a of the support leg 367 comes into contact with the flat surface 377a of the elevating member 377.
  • the support leg 365 rises along the shape of the upper surface of the elevating member 375 and comes into force contact with the plane 365a of the support leg 365 and the plane 375a of the elevating member 375.
  • Figure 3 0 (c) As shown, the part 353 that does not move up and down and all the area change parts 354, 355, 356, 357 are arranged on the same plane.
  • the area changing parts 354 to 357 can be controlled to move up and down by moving the height adjusting plate 370, so that a desired portion can be used as a liquid reservoir.
  • the shaking mechanism 291 of the shaking device 290 is installed as a pressing means at an upper position where the culture bag 242 of the culture bag tray 241 is arranged.
  • the shaking device 290 includes the shaking mechanism 291, the operating motor 292, the cam mechanism 293, and a positioning sensor (not shown).
  • an operating plate 291 a as a pressing means is movably arranged on the apparatus frame 250 via a guide rod 250 a, and a plurality of shaking plates 291 a are arranged on the bottom surface of the operating plate 291 a.
  • the protruding portion 291b is projected.
  • the operation plate 291a is alternately moved upward or downward by the operation of the cam mechanism 293 by the operation motor 292, so that the protrusion 291b of the operation plate 291a moves the culture bag 242 positioned below the shaking mechanism 291.
  • Repeated pressing, that is, pressing and releasing the culture bag 242 is repeated.
  • the medium in the culture bag 242 is agitated, the cells in the culture bag 242 float and move in the medium, and the cell distribution and oxygen concentration distribution in the culture bag 242 are made uniform, Cell proliferation is promoted.
  • the position of the operation plate 291a is detected by the positioning sensor and transmitted to the operation control PLC 223, and the measured value of the weigh scale 251 is transmitted to the operation motor 292 by the PLC 223. Be controlled.
  • the cell culture (shaking culture) in the culture bag 242 using the shaking device 290 may be performed before the culture bag and the cells are fully filled in the culture bag 242. It may be implemented later.
  • the shaking device 290 is controlled by the operation control PLC 223 based on the detection position of the working plate and the measurement value of the weighing scale 251.
  • an illumination lamp 301 is provided above the position where the portion of the culture bag 242 where the antibody is solidified is arranged, and the image acquisition means is provided below.
  • C CD camera 302 is installed.
  • a lens, a prism, a lens barrel, and other optical devices are additionally used according to the observation mode.
  • the illumination lamp 301 illuminates the culture bag 242 from above.
  • the CCD camera 302 displays the cells in the culture bag 242. The downward force is also photographed and the image is acquired.
  • the illumination operation of the illumination lamp 301 and the imaging operation of the CCD camera 302 are controlled by the operation control PLC 223, and an image of the cells in the culture bag 242 is acquired every predetermined time (for example, 6 hours).
  • the cell image at every predetermined time is stored in an image memory circuit (not shown) of the image processing unit 221.
  • the image processing unit 221 performs image processing, for example, binarization processing or multi-value processing, on a cell image every predetermined time stored in the image memory circuit of the image processing unit 221, and
  • the average value of the projected area of one cell and the increase rate of non-single cells, which are cell aggregates in which single cells are aggregated, are calculated as evaluation parameters for cell culture.
  • the average value of the projected area of a single cell is determined by attaching the culture bag 242 to the culture bag tray 241 and storing the culture bag tray 241 in the culture chamber 240. For example, the cell image power after 24 hours is calculated.
  • the force whether the decision is a non-single cell described above with reference to the can 100 mu m 2 projected area, to determine when a 100 m 2 or more and the non-single cell, the 100 m 2 or less It is judged as a single cell. This is because the projection area of a single cell at the initial stage of culture was measured and all were 100 m 2 or less. From the images of cells over time (for example, images after 24 hours, 48 hours, and 72 hours from the start of culture), the change in the ratio of non-single cells to total cells is calculated, and Calculate the rate of increase. The increase rate of the non-single cells and the average projected area of the single cells are output from the image processing unit 221 to the operation control PLC 223.
  • the PLC223 for operation control calculates the lag time for the average projected area force of a single cell, and estimates the growth start time of the cell.
  • the lag time is the time of the induction period required for inoculating cells into the antibody solid layer part of the culture bag tray 241 and starting the force growth.
  • the PLC223 for operation control determines the quality of the cell by judging the state of the cell culture from the start of cell growth, that is, whether the cell has the potential to grow by antibody stimulation. Based on this determination, the operation control PLC 223 stops culturing in the cell culture device 200 for cells with extremely low proliferation potential.
  • the PLC 223 for operation control calculates the minimum doubling time of the cell from the increase rate of the non-single cell.
  • the minimum fold time is the number of cells at a certain time. Is the time required to reach double the number of cells.
  • the operation control PLC 223 determines the timing of flow of the culture medium to the culture bag 242 and the flow acceleration by judging the culture state of the cell, that is, the proliferation ability of the cell from the minimum doubling time.
  • the operation control PLC 223 and the image processing unit 221 functioning as control means are a CPU that executes computation and control, a storage device (memory) that stores processing programs and data, and data. And an input / output circuit for connecting to an input device such as a keyboard, a mouse or a touch panel, and an output device such as a monitor.
  • the image processing unit 221 includes an image memory circuit that stores image data from the CCD camera 302.
  • an image of the cells in the culture bag 242 taken by the CCD camera 302 every predetermined time is subjected to image processing (for example, binarization processing or multi-value processing).
  • image processing for example, binarization processing or multi-value processing.
  • a program for calculating evaluation parameters of cell culture average projected area of single cells, increase rate of non-single cells) and the like are stored.
  • the storage device of the operation control PLC223 includes a program for determining the cell culture status (cell proliferation possibility, cell proliferation capability) from the cell culture evaluation parameters, and the cell culture status.
  • a program for controlling the devices for example, the supply pump 261, the discharge pump 271 and the like
  • the storage device of the operation control PLC 223 includes a device control device for controlling devices related to the culture device 210 and the culture unit 212 based on signals from various sensors of the cell culture device 200 and the culture unit 212.
  • the program is also stored.
  • the storage device of the image processing unit 221 also stores a device control program such as controlling the CCD camera 302 at predetermined time intervals to acquire cell images.
  • FIG. 31 and FIG. 32 show the antibody-stimulated intermittent perfusion culture process, in which cells are collected in the culture bag 242.
  • FIG. 31 the operator solidifies the antibody in the culture bag 242 in a clean bench or the like (W01 in FIG. 31), puts the medium, and incubates the culture bag 242 inoculated with the cells. Attach to Nourishing Bag Tray 241.
  • the operator attaches the culture medium bag 233 into which the culture medium has been placed in a clean bench or the like to the culture medium bag tray 231, attaches the waste liquid bag 234 to the waste liquid bag tray 232, and culture bag 242, the culture medium bag 233, and the waste liquid bag 234 and 234 are connected by a supply joint 264 and a waste liquid joint 274, respectively (W02 in FIG. 31).
  • the operator places the culture bag tray 241, medium bag tray 231 and waste solution bag tray 232 in the single culture unit 212 of the cell culture device 200 whose indicator light is lit in green, for example.
  • the culture bag tray 241 is supported by the frame 250 of the culture chamber 240, and the medium bag tray 231 and the waste solution bag tray 232 are installed in the low temperature chamber 230.
  • the operator connects the pump tubes 262 and 272 of the culture bag 242 to the supply pump 261 and the output pump 271 respectively (W03 in FIG. 31).
  • the operator confirms the image output of the CCD camera 302 in the culture bag 242 stored in the culture unit 212 (W05 in FIG. 31). Before confirming the image output, the operator activates the tilting motor 280 of the culture unit 212 to lower the area changing parts 2 52b and 252c of the mounting table 252 to form a liquid reservoir in the culture bag 242. (W04 in Fig. 31). Further, the operator measures the weight of the installed culture bag 242 with the weighing scale 251 of the culture unit 212.
  • the operator closes the culture unit door and starts cell culture in the culture unit 212 (W06 in Fig. 31).
  • the cells are stimulated by the antibody for growth in the liquid reservoir of the culture bag 242 (W07 in FIG. 31).
  • the CCD camera 302 of the culture unit 212 photographs the cells in the liquid reservoir of the culture bag 242 every predetermined time (for example, 6 hours), and the image processing unit 221 evaluates the cell culture from the captured image. Calculate the parameters.
  • the PLC 223 for operation control calculates the lag time based on the evaluation parameter, determines whether the cell is stimulated by the antibody and has a possibility of proliferation, and further calculates the minimum doubling time. Thus, the proliferation ability of the cell is determined (W08 in FIG. 31).
  • the PLC223 for operation control uses this cell when the cell is stimulated by the antibody in the culture bag 242 and no growth is observed after a certain period of time (eg, 24 hours).
  • the cell culture in the culture apparatus 200 is stopped (W08 ′ in FIG. 31).
  • the operation control PLC 223 determines that the cells in the culture bag 242 have the possibility of growth, it is based on the proliferation ability of the cells! To decide. Based on this determination, the operation control PLC 223 operates the supply pump 261 to feed the medium in the medium bag 233 of the medium bag tray 231 to the culture bag 242 (W09 in FIG. 31).
  • the PLC 223 for operation control determines whether or not the weight of the culture medium and the cells in the culture bag 242 measured by the weigh scale 251 exceeds the predetermined value b (W14 in FIG. 31).
  • Actuator motor 292 is started when the value reaches or exceeds the constant value b.
  • the shaking device 290 is activated, and shaking culture in which the shaking mechanism 291 of the shaking device 290 repeatedly presses the culture bag 242 is started (W15 in FIG. 31).
  • the operation control PLC 223 continues to determine whether the weight of the culture medium and cells in the culture bag 242 measured by the weigh scale 251 is equal to or greater than the predetermined value c (W16 in FIG. 32).
  • the supply pump 261 is stopped, the medium bag 233 force of the medium bag tray 231 also stops feeding of the medium to the culture bag 242 (W17 in FIG. 32), the operation motor 292 is stopped, and the culture is stopped. Stop shaking culture in bag 242 (W18 in Figure 32).
  • the PLC223 for operation control operates the discharge pump 271 after the cells settle in the culture bag 242 to remove the spent medium (the supernatant in the culture bag 242) from the waste bag tray 232 To the waste liquid bag 234 (W19 in Fig. 32). Thereafter, the PLC 223 for operation control uses the culture medium 242 in the culture bag 242 measured by the weighing scale 251. It is determined whether or not the weight is less than or equal to the predetermined value d (W20 in FIG. 32). When the weight is less than or equal to the predetermined value d, the discharge pump 271 is stopped to discharge the spent medium from the culture bag 242. Stop (W21 in Fig. 32).
  • the supply pump 261 is activated, the medium is fed from the medium bag 233 of the medium bag tray 231 to the culture bag 242, the operating motor 292 is activated, and the shaking culture is performed in the culture bag 242 by the shaking device 290 ( (W22 in Figure 32).
  • the operation control PLC 223 stops the supply pump 261 to stop the feeding of the medium from the medium bag 233 of the medium bag tray 231 to the culture bag 242 and performs shaking culture while the operation motor 292 is activated. Continue (W23 in Figure 32).
  • the PLC 223 for operation control determines whether or not a desired culture period depending on the use date and time of the cells to be propagated has been reached, or the image processing unit 221 determines whether the cells in the culture bag 242 are desired. It is determined whether or not the force has reached the number of cells (W24 in FIG. 32). When these culture periods or the number of cells have not been reached, the processing operations of steps W18 to W23 are repeated. When measuring the number of cells, stop shaking device 290 once and wait until the cells settle. Next, an image in the culture bag 242 is taken by the CCD camera 302. The image processing unit 221 estimates and calculates the number of cells present in the bag from the acquired image.
  • the above steps W18 to W23 are intermittent perfusion culture in which the spent medium in the culture bag 242 is discharged and the new medium is fed (fed) into the culture nodule 242. It is.
  • Step W24 the operation control PLC 223 stops the operation motor 292 as shown in FIG. 32, and stops the shaking culture in the culture bag 242. (W25 in Figure 32).
  • the discharge pump 271 is activated, and the spent medium in the culture bag 242 is discharged to the waste solution bag 234 in the waste solution bag tray 232, based on the measurement value of the weigh scale 251.
  • the cells are concentrated until the medium and cells in the culture bag 242 become approximately 1Z2 to 1Z3 (W26 in FIG. 32).
  • the PLC 223 for operation control then stops the discharge pump 271 and ends the cell culture (W27 in Fig. 32). After completion of the culture, the operator transfers the cells in the culture bag 242 to a centrifuge container in a clean bench or the like, and then the cells are circulated by centrifugation. (W28 in Figure 32).
  • FIGS. 33 and 34 show a case where the same antibody-stimulated intermittent perfusion culture process includes a process of collecting cells with a cell collection bag. Therefore, steps W31 to W56 in the steps shown in FIGS. 33 and 34 are the same as steps WO 1 to W26 in FIGS.
  • step W56 shown in FIG. 34 after the cells in the culture bag 242 are concentrated by starting the discharge pump 271, the operation control PLC 223 stops the discharge pump 271, and the waste bag 234 in the waste liquid tray 232 is removed. Prompt the operator to replace the cell collection bag (W57 in Figure 34).
  • This cell collection bag is a bag that can be attached to a centrifuge and used for centrifugation.
  • the operation control PLC 223 activates the discharge pump 271 and the operation motor 292.
  • the cells in the culture bag 242 are transferred together with the culture medium to the cell collection bag mounted on the waste bag tray 232 (W58 in FIG. 34).
  • the operation control PLC 223 stops the discharge pump 271 and the operating motor 292 to stop the cell recovery from the culture bag 242 and end the cell culture (W59 in FIG. 34).
  • the operator attaches the cell collection bag to the centrifuge and collects the cells by centrifugation (W60 in FIG. 34).
  • Steps X01 to X15 in the antibody-stimulated continuous perfusion culture process shown in FIG. 35 and FIG. 36 are the same as steps W01 to W15 in the antibody-stimulated intermittent perfusion culture process of FIG. 31 and FIG.
  • a filter (not shown) is disposed between the culture bag 242 and the discharge pump 271.
  • the PLC 223 for operation control is used while the culture medium is fed from the culture medium bag 233 of the culture medium bag tray 231 into the culture bag 242 and shaking culture is performed by the shaking device 290 in the culture bag 242.
  • the discharge pump 271 is activated and the culture bag 242 is used.
  • the medium is discharged into the waste bag 234 of the waste bag tray 232.
  • continuous perfusion culture in which the feeding of the medium to the culture bag 242 and the discharge of the medium from the culture bag 242 are simultaneously started in the culture bag 242 (XI 7 in FIG. 36).
  • the cells in the culture bag 242 are blocked from flowing by the filter and do not flow into the waste liquid bag 234.
  • shaking culture with a shaking device 290 is also performed at the same time.
  • the PLC 223 for operation control determines whether or not the desired culture period depending on the use date and time of the cells to be proliferated has been reached, or the image processing unit 221 determines whether the cells in the culture bag 242 are desired. Determine whether the force has reached the number of cells (X18 in Fig. 36). If these culture periods or cell numbers have not been reached, repeat the continuous perfusion culture in step X17. When measuring the number of cells, stop the shaking device 290 once and wait until the cells settle. Next, an image in the culture bag 242 is taken by the CCD camera 302. The image processing unit 221 estimates and calculates the number of cells present in the bag from the acquired image.
  • the operation control PLC 223 stops the perfusion culture and the shaking culture by stopping the supply pump 261, the discharge pump 271 and the operation motor 292 when the desired culture period or cell number in step X18 is reached ( XI 9 in Figure 36).
  • the PLC 223 for operation control activates the discharge pump 271 after the cells settle in the culture node 242 to discharge the spent medium in the culture bag 242 to the waste bag 234 in the waste bag tray 232, and the weighing scale 251 Based on the measured value, concentrate the cells until the culture medium and cells in culture bag 242 are approximately 1Z2 to 1Z3 (X20 in Fig. 36).
  • the reason why the shaking device is stopped during the concentration process is to prevent a large amount of cells from flowing into the tube and clogging the filter.
  • the operation control PLC 223 then stops the discharge pump 271 and ends the cell culture (X21 in Fig. 36). After completion of the culture, the operator moves the cells in the culture bag 242 to a centrifuge container in a clean bench or the like, and then collects the cells by centrifugation (X22 in FIG. 36).
  • Steps YO 1 to Y15 in the antibody-stimulated continuous perfusion culture process shown in FIGS. 37 and 38 are the same as those in the antibody-stimulated intermittent perfusion culture process shown in FIGS. Since it is the same as steps WO 1 to W15, description thereof is omitted.
  • the PLC 223 for operation control determines whether the weight of the medium and cells in the culture bag 242 measured by the weighing scale 251 is equal to or greater than the predetermined value c (Y16 in FIG. 38). At this point, the supply pump 261 is stopped, the medium bag 233 force of the medium bag tray 231 is also stopped, and the feeding of the medium to the culture bag 242 is stopped, and the shaking culture is continued with the operating motor 292 activated (Fig. 38 Y17).
  • the PLC 223 for operation control determines whether or not the desired culture period depending on the use date and time of the cells to be proliferated has been reached, or the image processing unit 221 determines whether the cells in the culture bag 242 are desired. It is determined whether or not the force has reached the cell number (Y18 in FIG. 38). If the culture period or cell number has been reached, shaking culture is continued (No in Y18).
  • the image processing unit 221 estimates and calculates the number of cells present in the bag from the acquired image.
  • the operation control PLC 223 stops the operation motor 292 as shown in Fig. 38 to stop the shaking culture in the culture bag 242. (Y19 in FIG. 38), after the cells settle in the culture bag 242, the discharge pump 271 is activated to discharge the spent medium in the culture bag 242 to the waste bag 234 in the waste bag tray 232 and weigh Based on the 251 measurements, the cells are concentrated until the medium and cells in the culture bag 242 are approximately 1-2 cm to 1-3 cells (Fig. 38, ⁇ 20).
  • the operation control PLC 223 then stops the discharge pump 271 to end the cell culture ((21) in Fig. 38). After completion of the culture, the operator moves the cells in the culture bag 242 to a centrifuge container in a clean bench or the like, and then collects the cells by centrifugation ( ⁇ 22 in FIG. 38).
  • FIG. 39 and FIG. 40 showing this processing operation in the case where the same antibody-stimulated simple fed-batch culture process includes a process of recovering cells using a cell recovery bag is shown in FIGS. Accordingly, steps ⁇ ⁇ ⁇ ⁇ 31 to ⁇ 50 of the process shown in FIGS. 39 and 40 are the same as steps ⁇ 1 to ⁇ 20 of FIGS.
  • step ⁇ 50 shown in Fig. 40 the culture bag 242 is activated by the activation of the discharge pump 271.
  • the operation control PLC 223 stops the discharge pump 271 and prompts the operator to replace the waste bag 234 of the waste liquid tray 232 with a cell collection bag (Y51 in FIG. 40).
  • This cell collection bag is a bag that can be attached to a centrifuge and used for centrifugation.
  • the operation control PLC 223 activates the discharge pump 271 and the operating motor 292, and shakes the culture bag 242 with the shaking device 290. Then, the cells in the culture bag 242 are transferred together with the medium to the cell collection bag attached to the waste bag tray 232 ((52 in FIG. 40). Then, the operation control PLC 223 stops the first discharge pump 271 and the operating motor 292 to stop the cell recovery from the culture bag 242 and terminate the cell culture ((53 in FIG. 40). After the cell recovery is completed, the operator attaches the cell recovery bag to the centrifuge, and the cells are recovered by centrifugation ((54) in FIG. 40).
  • Image of cell in culture bag 242 captured by CCD camera 302 is processed by image processing unit 221 to evaluate cell culture evaluation parameters (average projected area of single cell, increase of non-single cell) Speed), and the operation control PLC223 evaluates and evaluates the culture status of the cell (proliferation potential and proliferation capability of the cell), and the culture operation according to this culture status (predetermined from the medium bag 233 to the culture bag 242) Perform medium feeding and timing at flow acceleration.
  • cell culture evaluation parameters average projected area of single cell, increase of non-single cell
  • Speed the operation control PLC223 evaluates and evaluates the culture status of the cell (proliferation potential and proliferation capability of the cell), and the culture operation according to this culture status (predetermined from the medium bag 233 to the culture bag 242) Perform medium feeding and timing at flow acceleration.
  • cells from one patient can be inoculated into a culture bag 242 stored in a single culture unit 212, and each cell can be cultured according to the culture status of the cell, thus realizing an appropriate culture operation. it can.
  • the culturing operation can be performed in units of hours, and the culturing can be promoted to shorten the culturing period.
  • FIG. 1 is a configuration diagram showing a first embodiment of a cell culture device according to the present invention.
  • FIG. 2 is a perspective view showing the thermostatic chamber of FIG.
  • FIG. 3 is a perspective view showing a culture cassette housed in a canister in the thermostatic chamber of FIG. 1.
  • FIG. 4 is a perspective view showing the culture bag tray, medium cassette and cell inoculation cassette in FIG. 3, respectively.
  • FIG. 5 is a layout diagram showing a configuration of a culture unit including one caster in the thermostatic chamber of FIG. 2 and a culture cassette housed in the caster.
  • FIG. 6 is a block diagram showing a control system of the culture unit of FIG.
  • FIG. 5 The shaking mechanism of the shaking device of FIG. 5 is shown, (A) is a perspective view, and (B) is a side view.
  • FIG. 8 is a process diagram showing a culture procedure in the culture unit of FIG. 5.
  • FIG. 9 shows a culturing procedure in the culturing unit of FIG. 5, and is a process diagram following FIG. 8.
  • FIG. 10 shows a culture procedure in the culture unit of FIG. 5, and is a process diagram following FIG.
  • FIG. 11 is a process chart subsequent to FIG. 10, showing the culture procedure in the culture unit of FIG. [12]
  • FIG. 12 is a flowchart showing the processing operation of the induction factor stimulation intermittent perfusion culture process in the culture unit of FIG.
  • FIG. 13 is a flowchart showing a continuation of the processing operation of FIG.
  • FIG. 14 is a flowchart showing the processing operation of the inducing factor-stimulated intermittent perfusion culture process (including cell recovery processing by the cell recovery bag) in the culture unit of FIG.
  • FIG. 15 is a flowchart showing a continuation of the processing operation of FIG.
  • FIG. 16 is a flowchart showing the processing operation of the induction factor stimulation continuous perfusion culture process in the culture unit of FIG.
  • FIG. 17 is a flowchart showing a continuation of the processing operation of FIG.
  • FIG. 18 is a diagram showing a flow of cell culture-related data before cell culture in the cell culture system of FIG.
  • FIG. 19 is a diagram showing a flow of cell culture-related data during cell culture of the cell culture system of FIG. 1.
  • FIG. 20 is a diagram showing a flow of cell culture-related data after cell culture in the cell culture system of FIG. 1.
  • ⁇ 21 A layout diagram showing the configuration (differentiation induction state) of the culture unit in the second embodiment of the cell culture device according to the present invention.
  • FIG. 22 is a layout diagram showing the configuration of the culture unit of FIG.
  • FIG. 23 is a flowchart showing the processing operation of the split induction-induced intermittent perfusion culture process in the culture unit of FIGS. 21 and 22.
  • FIG. 24 is a flowchart showing a continuation of the processing operation of FIG.
  • FIG. 25 is a configuration diagram showing a third embodiment of the cell culture system according to the present invention.
  • FIG. 26 is a configuration diagram showing a fourth embodiment of the cell culture device according to the present invention.
  • FIG. 28 is a perspective view showing a configuration of the mounting table in FIG. 27.
  • FIG. 29 A plan view showing a configuration of a mounting table according to a modification of the fourth embodiment, wherein (a) is a diagram showing a state in which the parts that do not move up and down and all the area changing parts are on the same plane b) is a diagram showing a state in which the area changing part is lowered.
  • FIG.30 A partial cross-sectional view along the ⁇ - ⁇ line in Fig. 29 (a).
  • (B) is a diagram showing a state where three of the four area-changing parts are on the same plane
  • (c) is a figure showing all the parts not moving up and down It is a figure which shows the state which has an area change part on the same plane.
  • FIG. 31 is a flowchart showing a processing operation in the case where the culture bag includes a process of collecting cells in the antibody-stimulated intermittent perfusion culture process in the culture unit of FIG. 27.
  • FIG. 32 is a flowchart showing a continuation of the processing operation of FIG. 31.
  • FIG. 33 is a flowchart showing the processing operation in the case where the cell recovery bag includes a process of recovering cells over the antibody-stimulated intermittent perfusion culture process in the culture unit of FIG. 27.
  • FIG. 34 is a flowchart showing a continuation of the processing operation of FIG. 33.
  • FIG. 28 is a flowchart showing the processing operation of the antibody-stimulated continuous perfusion culture process in the culture unit of FIG.
  • FIG. 36 is a flowchart showing a continuation of the processing operation of FIG. 35.
  • FIG. 37 is a flowchart showing the processing operation of the antibody-stimulated simple fed-batch culture step in the culture unit of FIG. 27.
  • FIG. 38 is a flowchart showing a continuation of the processing operation of FIG. 37.
  • FIG. 39 is a flowchart showing a processing operation in the case where the antibody-stimulated simple fed-batch culture step in the culture unit of FIG.
  • FIG. 40 is a flowchart showing a continuation of the processing operation of FIG. 39.
  • Waste liquid bag (Waste liquid storage means, used medium storage container)

Abstract

La présente invention concerne un appareil de culture cellulaire permettant de mener une culture cellulaire respectant les états de culture cellulaire tout en demandant moins de l'opérateur. L'appareil de l'invention est ainsi constitué de plusieurs éléments. Ainsi, un sac de culture (18) sert à faire proliférer les cellules. Une cassette d'inoculation des cellules (19) permet de stimuler les cellules avec un inducteur de prolifération. A la place, on peut avoir un sac de culture (242) servant de récipient de culture pour la stimulation d'anticorps et la prolifération. Une cassette de milieu de culture (20) permet de conserver un milieu alimentant le sac de culture (18) et la cassette d'inoculation des cellules (19). Une caméra électronique (88) donne une image des cellules dans la cassette d'inoculation des cellules. Enfin, un ordinateur de traitement d'image ainsi qu'un ordinateur de régulation de fonctionnement permettent, d'apprécier les états de culture des cellules (proliférabilité et aptitude à la prolifération des cellules) sur la base de l'image des cellules donnée par la caméra électronique. Ces ordinateurs permettent par conséquent de mener la culture sur la base de cette appréciation.
PCT/JP2006/321901 2005-11-01 2006-11-01 Appareil, procede, logiciel et systeme de culture cellulaire WO2007052716A1 (fr)

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AU2006309682A AU2006309682A1 (en) 2005-11-01 2006-11-01 Cell culture apparatus, cell culture method, cell culture program and cell culture system
JP2007542788A JP5243038B2 (ja) 2005-11-01 2006-11-01 細胞培養装置
EP06822821.2A EP1944358B1 (fr) 2005-11-01 2006-11-01 Appareil, procede, logiciel et systeme de culture cellulaire
US12/084,363 US8383395B2 (en) 2005-11-01 2006-11-01 Cell culture apparatus, cell culture method, cell culture program and cell culture system

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EP1944358A1 (fr) 2008-07-16
JP5243038B2 (ja) 2013-07-24
KR20080072022A (ko) 2008-08-05
AU2006309682A1 (en) 2007-05-10
JPWO2007052716A1 (ja) 2009-04-30
EP1944358B1 (fr) 2017-08-02
US20090042293A1 (en) 2009-02-12

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